JP2018050550A - Spinner culture device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a spinner culture device which can culture cells in an optimum state by acquiring the state of the cells during agitation and controlling the agitation.SOLUTION: The spinner culture device comprises: a culture tank 2 in which a culture solution including cells are contained; an agitation means 5 which agitates the culture solution in the culture tank 2; an acquisition means 10 which acquires cell data of cells in the culture solution 2; and a control means 11 which controls the agitation means according to the state of the cells acquired from the cell data acquired from the acquisition means 10.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a culture apparatus for culturing a part of an animal or plant cell / tissue, or an individual, a microorganism, a fungus body, or the like, particularly cells such as stem cells and animal cells.

  Conventionally, a culture solution containing cells in a culture tank is stirred by rotating a stirring blade (rotary blade) to culture cells in the culture tank. There is a reciprocating blade type stirring and culturing apparatus for moving and stirring to culture cells in the culture tank.

  FIG. 13 shows the conventional reciprocating blade type agitation culture apparatus 1, and reference numeral 2 denotes a culture tank of the agitation culture apparatus 1, which has a cylindrical body, for example, a stem cell or an animal cell. Contains cells and culture medium.

  Reference numeral 3 denotes a drive shaft, which is inserted into the culture tank 2 through the central opening 4 of the ceiling 2a of the culture tank 2.

  In addition, an upper and lower stirring blade 5 is connected and fixed to the lower end of the drive shaft 3 so as to be orthogonal to the drive shaft 3, and the upper and lower stirring blade 5 has, for example, a plate shape having a major axis and a minor axis. It consists of a body and is formed in, for example, a rectangle, an ellipse, or an oval.

  In addition, a deformable bellows 6 is provided between the drive shaft 3 and the edge of the opening 4, and the culture tank 2 is hermetically sealed from the outside by the bellows 6, and the drive shaft 3 can be moved up and down.

  Reference numeral 7 denotes a reciprocating drive device connected to the upper end of the drive shaft 3 and provided above the ceiling portion 2a. The upper and lower stirring blades 5 reciprocate up and down by driving the reciprocating drive device 7. Formed as follows.

  The reciprocating drive device 7 is supported by a support device (not shown).

  Reference numeral 8 denotes a control device for the reciprocating drive device 7. The control device 8 drives the reciprocating drive device 7 so as not to damage the cells and to achieve appropriate mixing uniformity. The upper and lower stirring blades 5 were moved up and down to stir and culture the culture solution containing the cells contained in the culture tank 2.

  Reference numeral 7 a denotes a driver that receives the control signal from the control device 8 and controls the operation of the reciprocating drive device 7.

  For example, Patent Document 1 is an example of the reciprocating blade type stirring culture apparatus.

Japanese Patent Application Laid-Open No. 2004-243161

  In the conventional reciprocating blade type agitating culture apparatus, in order to stir the inside of the tank so as to reduce the shearing force so as not to damage the cells and to improve the mixing uniformity in the tank, Based on the flow characteristics of the liquid, the vertical agitating blade 5 is reciprocated up and down by determining the vertical amplitude of the upper and lower agitating blade 5 and the frequency of the reciprocating vibration based on, for example, a sine curve.

  Also, in the conventional rotary blade type agitation culture apparatus, in order to stir the inside of the tank mainly to improve the mixing uniformity in the tank, the rotational speed of the rotary blade is determined and the rotary blade is rotated. I was letting.

  However, in any of the conventional agitation culture apparatuses described above, the agitation is performed simply by culturing the cells, considering only the flow characteristics in the tank, and the cell concentration, cell diameter, cell circularity, cell Cell status such as diameter standard deviation was not considered.

  The present inventor has shown that the rotational speed of the rotor blade (for example, the speed of the rotor blade during a desired time. It is found that the state of the cells such as the cell concentration in the culture tank can be adjusted (controlled) by controlling such as changing / maintaining, and if it is a reciprocating blade type stirring culture apparatus, By controlling the amplitude, speed, and acceleration of the upper and lower stirring blades (or changing the amplitude, speed, and acceleration) (or the amplitude, frequency, and waveform (representing the speed of the upper and lower stirring blades during a desired time). The average cells in the culture tank by changing or maintaining the speed of the upper and lower stirring blades during one period of vibration, for example, sine wave, rectangular wave, triangular wave, etc. Diameter, average circularity of cells, cells It was found that the state of the cells such as the standard deviation of the cell can be adjusted (controlled), and further, the state of the cells in the culture was measured during the agitation culture, and depending on the measured value, Control one of the amplitude, speed, acceleration (or amplitude, frequency, waveform) of the agitating blade, or a combination of these, or change / maintain all of them to control the cells in the optimal state. It was found that it can be cultured.

  In order to achieve the above object, the stirring culture apparatus of the present application includes a culture tank in which a culture solution containing cells is stored, a stirring means for stirring the culture solution in the culture tank, and cell data of cells in the culture solution. And a control means for controlling the stirring means in accordance with the state of the cells obtained from the cell data obtained by the obtaining means.

  Further, the acquisition means includes imaging means for imaging a culture solution containing cells in the culture tank or imaging a culture solution containing cells in the culture tank extracted outside the culture tank. It is characterized by that.

  Further, the acquisition means is a measuring device that acquires cell data of cells in the culture tank or cell data of cells in the culture tank extracted from the culture tank.

  The control means comprises an arithmetic storage processing device, the arithmetic storage processing device comprising a storage unit, a measurement unit for measuring a cell state from the cell data obtained by the acquisition unit, and the measured cell And a control unit that controls the agitation means based on the result of the comparison unit. .

  The cell state is at least one of cell concentration, average cell diameter, average circularity of cells, or standard deviation of cell diameter.

  The stirring means is a means for reciprocating the stirring blades or a means for rotating the stirring blades.

  The stirring blade is formed in a rectangular shape, an elliptical shape, or an oval shape.

  An agitating blade inserted into a stirring tank having a cylindrical body and connected and fixed so as to be orthogonal to the lower end surface portion of the reciprocating drive shaft is an elliptical plate having a major axis and a minor axis passing through the center. In the stirring blade, in the stirring blade, a portion separated by a long diameter line and a short diameter line passing through the center thereof, a pair of diagonal portions that are not adjacent to each other are bent obliquely upward along the long diameter line, or A pair of diagonal portions that are not adjacent to each other is formed by bending in a diagonally downward direction along the major axis.

  An agitating blade inserted into a stirring tank having a cylindrical body and connected and fixed so as to be orthogonal to the lower end surface portion of the reciprocating drive shaft is an elliptical plate having a major axis and a minor axis passing through the center. The stirrer blade is formed by bending two pairs of diagonal portions that are not adjacent to each other at a portion separated by a long diameter line and a short diameter line passing through the center of the stirring blade in a diagonally upward or downward direction along the long diameter line, respectively. The portions adjacent to each other with the long diameter wire or the short diameter wire projecting in opposite directions above and below are formed.

  In addition, an automatic additive addition device is provided.

  The culture stirring method of the present invention includes a first step of stirring a culture solution containing cells in a culture tank by a stirring means, a second step of acquiring cell data of cells in the culture tank, and the cell data. According to the state of the obtained cell, the stirring control means comprises a third step for controlling the stirring, and if necessary, a fourth step for repeating the second step and the third step. To do.

  ADVANTAGE OF THE INVENTION According to this invention, while maintaining the mixing uniformity in a culture tank, a shearing effect is suppressed, and the state of cells can be adjusted (controlled) to a desired state, and can be cultured early in cells in an optimal state. It becomes like this.

It is a partial vertical side view of the stirring culture apparatus of the present invention. It is a partial longitudinal cross-sectional side view of the other Example of the stirring culture apparatus of this invention. It is a partial longitudinal cross-sectional side view of other Example of the stirring culture apparatus of this invention. It is a partial longitudinal cross-sectional side view of other Example of the stirring culture apparatus of this invention. It is a top view of the stirring blade of the stirring culture apparatus of this invention. It is a top view of the other Example of the stirring blade of the stirring culture apparatus of this invention. It is a top view of other Example of the stirring blade of the stirring culture apparatus of this invention. It is a X direction arrow line view in FIG. It is a Y direction arrow directional view in FIG. It is a top view of other Example of the stirring blade of the stirring culture apparatus of this invention. It is a X direction arrow directional view in FIG. It is a Y direction arrow directional view in FIG. It is a partial vertical side view of the conventional stirring culture apparatus.

  The example of the form for carrying out the present invention is shown below.

  A first embodiment of the present invention will be described with reference to FIGS. In addition, the same code | symbol as the past is attached | subjected to the same code | symbol, and description is abbreviate | omitted.

  FIG. 1 shows a reciprocating blade type stirring culture apparatus 9 of the present invention. The reciprocating blade agitation culture apparatus 9 obtains cell data as a basis for measuring (calculating) the state of the cells from the cells in the culture tank 2 in addition to the conventional reciprocating blade agitation culture apparatus 1. And measuring (calculating) the state of the cells in the culture tank 2 such as the cell concentration and the average cell diameter from the acquisition means 10 and the cell data obtained from the acquisition means 10, and based on the result of the measurement (calculation), Control means for controlling the amplitude, speed, acceleration (or amplitude, frequency, waveform) of the upper and lower agitating blades 5 of the reciprocating drive device 7 or changing or maintaining all of them in combination. 11 is provided.

  Examples of the cell data include an image obtained by imaging cells in a culture solution, individual cell diameters, circularity of individual cells, cell diameter distribution, and cell concentration.

  Examples of the cell state include cell concentration, average cell diameter, average circularity of cells, standard deviation of cell diameter, and the like.

  As the acquisition means 10, for example, as shown in FIG. 1, as the acquisition means 10 a, outside the culture tank 2 for storing a culture solution (including cells; the same applies hereinafter) in the culture tank 2. The dedicated sampling container 12 provided and the culture medium in the culture tank 2 are extracted into the sampling container 12 by extraction means (not shown) such as a pump, and provided through the ceiling portion 2a of the culture tank 2. The sampling tube 13 and the imaging means 14 such as a camera for imaging the cells in the culture solution stored in the sampling container 12.

  The control means 11 includes an arithmetic storage processing device 15 such as a personal computer, for example. The arithmetic storage processing device 15 is a cell such as an image obtained by the arithmetic processing portion 15a, the storage portion 15b, and the acquisition means 10. The measurement unit 15c that measures (calculates) the state of the cell such as the cell concentration, the average cell diameter, the average circularity of the cell, and the standard deviation of the cell diameter by performing, for example, image processing and various calculations using the data. And a comparison unit 15d that compares the measured state of the cell with the state of the target cell, and based on the result of this comparison, the reciprocating drive device 7 is driven, and the amplitude of the upper and lower stirring blades 5 is , A drive unit 15e for controlling and changing and maintaining speed, acceleration (or amplitude, frequency, waveform), and an external output unit 15f for displaying various information on a monitor (not shown).

  The target cell state is stored in advance in the storage unit 15b of the arithmetic storage processing device 15.

  Next, the operation and effect of the stirring culture apparatus of the present invention will be described.

  First, the culture solution containing the cells is placed in the culture tank 2, and the control means 11 causes the upper and lower stirring blades 5 to be sine waves, for example, so as to improve the mixing uniformity without damaging the cells. In the vibration based on this, the culture solution in the culture vessel 2 is stirred by moving up and down with a desired amplitude and a desired frequency.

  Next, after a desired time has elapsed, a desired amount of the culture solution in the culture tank 2 being stirred is stored in the sampling vessel 12 through the sampling tube 13, and the stored culture solution is stored in the camera. The cell data including the image obtained by the imaging unit 14 is stored in the storage unit 15 b of the arithmetic storage processing device 15.

  Next, the cell data composed of the images stored in the storage unit 15b is processed by the measurement unit 15c, and the cell concentration, the average cell diameter, the average circularity of the cells, the standard deviation of the cell diameter, etc. The state of the cell is measured and stored in the storage unit 15b.

  Next, the comparison unit 15c compares the stored measured cell state with the target cell state stored in the storage unit 15c in advance, and according to the comparison result, For example, if the state of the target cell is different, the drive unit 15e changes the amplitude and frequency of the upper and lower stirring blades 5 by the drive unit 15e so that the target cell state is obtained. To drive.

  For example, when it is desired to adjust so as to increase the average cell diameter, for example, the frequency is changed so that the frequency of the upper and lower stirring blades 5 of the reciprocating drive device 7 is increased.

  In addition, for example, when it is desired to adjust the cell concentration to increase, the amplitude, frequency, and waveform of the upper and lower stirring blades 5 are adjusted so that a shearing force corresponding to the characteristics of the cell is generated.

  For example, when it is desired to increase the standard deviation of the cell diameter, the frequency of the upper and lower stirring blades 5 is adjusted to be within a desired range, for example, 1.2 to 3.05 Hz.

  The control of stirring for adjusting the state of the cells, that is, control of the amplitude, frequency, and waveform (or amplitude, speed, and acceleration. The same applies hereinafter) of stirring is an example, and the average cell diameter Amplitude, frequency, and waveform control to control cell, amplitude, frequency, and waveform control to control average circularity of cells, and amplitude, frequency, and waveform control to control standard deviation of cell diameter Since it differs depending on the installation conditions such as the shape of the cell tank and stirring blade, and the culture conditions such as the type of cells and culture medium, the amplitude, frequency, and waveform should be controlled according to the corresponding conditions. .

  Next, after the lapse of a desired time, the cell data in the culture tank 2 during stirring is obtained as described above, the state of the cell is measured, and compared with the state of the target cell. If the state is different from the cell state, the amplitude and frequency of the upper and lower agitating blades 5 are changed again by the drive unit 11e to drive the reciprocating drive device 7, and the culture solution in the culture tank 2 is removed. Allow to stir.

  Such an operation is performed every desired time during stirring culture.

  Note that cell data may be acquired and controlled in real time.

  In addition, when the cell data obtained by the acquisition means is a cell state, the cell data may be used as the cell state and compared with the target cell state.

  Further, in the agitation culture, for example, it is free to appropriately stop the agitation and restart the agitation again.

  ADVANTAGE OF THE INVENTION According to this invention, while maintaining the mixing uniformity in a culture tank, a shearing effect is suppressed, and the state of cells can be adjusted (controlled) to a desired state, and can be cultured early in cells in an optimal state. It becomes like this.

  In addition, even if the difference in the mixing tank, the scale, etc., the culture conditions are different, and the amplitude and frequency conditions of the upper and lower stirring blades are unknown, the amplitude, frequency and waveform of the upper and lower stirring blades will be corrected. As a result, the cells can be cultured in an optimal state.

  Further, the culture tank 2 is further provided with an additive automatic addition device (not shown) that can automatically add additives such as chemicals and oxygen, depending on the state of cells such as average cell diameter and cell concentration. The additive may be added automatically.

  In addition, the stirring tank 2 is provided with a dissolved oxygen amount sensor, a PH sensor, etc., and depending on the state of the cell, the dissolved oxygen amount, the PH value, etc., the dissolved oxygen amount, the PH value, etc. May be adjusted automatically.

  As shown in FIG. 2, in the acquisition means 10a, the sampling container 12 and the sampling tube 13 are omitted, and the cells in the culture medium in the culture container 2 are directly imaged by the imaging means 14. (Acquisition means 10b).

  Reference numeral 16 denotes a light source.

  Further, as shown in FIG. 3, in the acquisition means 10a, instead of providing the sampling container 12 and the imaging device 14, a measurement such as an ultrasonic particle size distribution measuring device is provided in the flow path in the middle of the sampling tube 13. An apparatus 17 is provided to acquire cell data such as cell diameter and cell distribution of individual cells flowing through the sampling tube 13 using the measurement apparatus 17 (acquisition means 10c), and the measurement unit 15c Thus, the state of the cell may be measured from the cell data.

  Further, as shown in FIG. 4, in the acquisition means 10a, instead of the sampling vessel 12, the sampling tube 13, and the imaging means 14, the ceiling portion 2a of the culture tank 2 is penetrated, and the tip portion thereof is used as the culture portion. Another measuring device 18 comprising an inline sensor type particle size distribution / shape analysis sensor based on, for example, FBRM (focused beam reflection measurement method) technology provided in the culture solution in the tank 2 may be provided (acquiring means 10d). ).

  Then, using the other measuring device 18, cell data such as cell diameter, circularity, cell distribution, etc. of individual cells in the culture solution 2 are acquired, and the cell is measured by the measuring unit 15c. You may make it measure the state of a cell from data.

  In addition, when the cell data acquired by the measurement device 17 or another measurement device 18 is in a cell state (that is, when the cell state can be directly acquired by the device), the acquisition is performed. The obtained cell data may be used as a cell state to be compared with a target cell state.

  The blade diameter of the upper and lower agitating blades 5 is such that the distance A between the blade tip of one side where the upper and lower agitating blades are the smallest and the stirring vessel is the diameter on the other side perpendicular to the diameter of the one side. This is formed to be longer than the distance B between the tip of the blade and the stirring tank, and the space of the distance A is formed so as to ensure a liquid flow path when the upper and lower stirring blades 5 move up and down.

  For example, the stirring diameter of the upper and lower stirring blades 5 is 30% or more and less than 100% of the culture tank diameter.

  For example, when the cross section of the culture tank 2 is circular, the upper and lower stirring blades 5 are, as shown in FIG. 5, an elliptical or oval plate having a major axis 5a and a minor axis 5b. It consists of a body 19.

  Instead of the plate-like body 19, as shown in FIG. 6, a horizontally long plate-like body 20 formed by cutting the top and bottom of a disk blade or a vertical stirring blade made of a rectangular plate-like body may be used. Good.

  Further, instead of the upper and lower stirring blades 5, as shown in FIGS. 7 to 10, for example, a long-diameter line 21 a and a short-diameter line 21 b are formed of a plate-like body formed in an elliptical shape and pass through the center of the elliptical shape. The upper and lower sides having the inclined blade portions 22a and 22a are formed by making a cut along the short diameter line 21b and bending a pair of diagonal parts not adjacent to each other diagonally upward along the long diameter line 21a. A stirring blade 21 may be used.

  The bending angle of the inclined blade portion 22a is, for example, 30 degrees.

  Further, instead of the vertical motion agitating blade 5, as shown in FIGS. 8 to 10, for example, it is composed of a plate-like body formed in an elliptical shape, and a major axis 23a and a minor axis 23b passing through the center of the ellipse. In the portion delimited by, and cut along the minor diameter line 23b, and bend two pairs of diagonal portions that are not adjacent to each other obliquely upward or obliquely downward along the major diameter line 23a, respectively. You may use the upper and lower stirring blade 23 which has the inclined blade | wing part 24a, 24b which the part which adjoins across the wire | line 23a or the short diameter wire | line 23b protruded in the mutually reverse direction of the upper direction and the downward direction.

  Although the said Example showed the case of the reciprocating stirring culture apparatus, it is applicable also to a rotary blade type stirring culture apparatus.

  For example, as agitation control by rotation, the rotation speed of the rotor blade (for example, one representing the speed during one rotation) is changed.

  In the rotary blade culture apparatus, for example, when it is desired to increase the cell concentration, the cell concentration is adjusted by increasing the number of rotations, for example.

  The cell culture device of the present invention is used for a culture device in medical products, foods and the like.

DESCRIPTION OF SYMBOLS 1 Reciprocating blade type stirring culture apparatus 2 Culture tank 2a Ceiling part 3 Drive shaft 4 Opening part 5 Vertical stirring blade 5a Long diameter 5b Short diameter 6 Bellows 7 Reciprocating drive apparatus 7a Driver 8 Control apparatus 9 Reciprocating blade type stirring culture apparatus 10 Acquisition Means 10a Acquiring means 10b Acquiring means 10c Acquiring means 10d Acquiring means 11 Control means 12 Sampling container 13 Sampling tube 14 Imaging means 15 Arithmetic storage processing device 15a Arithmetic processing section 15b Storage section 15c Measuring section 15d Comparison section 15e Driving section 15f External output section 16 Light source 17 Measuring device 18 Measuring device 19 Plate-like body 20 Plate-like body 21 Vertical stirring blade 21a Long diameter wire 21b Short diameter wire 22a Inclined blade 23 Vertical stirring blade 23a Long diameter wire 23b Short diameter wire 24a Inclined blade 24b Inclined blade

Claims (12)

A culture vessel containing a culture medium containing cells;
Stirring means for stirring the culture solution in the culture tank;
Obtaining means for obtaining cell data of cells in the culture solution;
An agitation culture apparatus comprising: a control unit that controls the agitation unit in accordance with a cell state obtained from the cell data obtained by the acquisition unit.   The acquisition means comprises imaging means for imaging a culture solution containing cells in the culture tank, or imaging a culture solution containing cells in the culture tank extracted outside the culture tank. The stirred culture apparatus according to claim 1, wherein   The said acquisition means is a measuring apparatus which acquires the cell data of the cell in the said culture tank which extracted the cell data of the cell in the said culture tank, or the outside of this culture tank. The stirring culture apparatus described in 1.   The control means includes an arithmetic storage processing device, the arithmetic storage processing device includes a storage unit, a measurement unit for measuring a cell state from the cell data obtained by the acquisition unit, and the measured cell state And a comparison unit that compares the state of the target cell stored in the storage unit, and a control unit that controls the agitation means based on a result of the comparison unit. The stirring culture apparatus described in any one of 1 to 3.   5. The cell state according to claim 1, wherein the cell state is at least one of a cell concentration, an average cell diameter, an average circularity of the cells, or a standard deviation of the cell diameter. The stirring culture apparatus described in 1.   The agitation culture apparatus according to any one of claims 1 to 5, wherein the agitation means is a means for reciprocating an agitation blade.   The agitation culture apparatus according to any one of claims 1 to 5, wherein the agitation means is a means for rotating an agitation blade.   The stirring culture apparatus according to claim 6, wherein the stirring blade is formed in a rectangular shape, an elliptical shape, or an oval shape. An agitating blade inserted into a stirring tank having a cylindrical body and connected and fixed so as to be orthogonal to the lower end surface portion of the reciprocating drive shaft is an elliptical plate-like body having a major axis and a minor axis passing through the center. Consists of
In the agitating blade, a pair of diagonal portions that are not adjacent to each other are separated by a long diameter line and a short diameter line that pass through the center of the stirring blade, and a pair of diagonal portions that are not adjacent to each other are bent obliquely upward along the long diameter line, or are not adjacent to each other. 7. The agitation culture apparatus according to claim 6, wherein a pair of portions are formed by bending in a diagonally downward direction along the major axis. An agitating blade inserted into a stirring tank having a cylindrical body and connected and fixed so as to be orthogonal to the lower end surface portion of the reciprocating drive shaft is an elliptical plate-like body having a major axis and a minor axis passing through the center. Consists of
In the stirring blade, in a portion separated by a long diameter line and a short diameter line passing through the center thereof, two pairs of diagonal lines that are not adjacent to each other are bent obliquely upward or diagonally downward along the long diameter line, respectively. The agitation culture apparatus according to claim 6, wherein adjacent portions across the minor axis are formed to protrude upward and downward in opposite directions.   The agitation culture apparatus according to any one of claims 1 to 10, wherein an automatic additive addition apparatus is provided. A first step of stirring the culture solution containing cells in the culture tank by a stirring means;
A second step of acquiring cell data of cells in the culture tank;
A third step of controlling the stirring by stirring control means according to the state of the cells obtained from the cell data;
A culture stirring method comprising the fourth step of repeating the second step and the third step as necessary.

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