Abstract:
A culture system comprises: a preparatory culture vessel and a main culture vessel that accommodate cells and a solution; a main stage that holds the preparatory culture vessel and the main culture vessel; a connecting tube that connects the culture vessels; a valve that opens and closes the connecting tube; and a rotating mechanism that rotates the main stage and imparts a height difference between the culture vessels to transfer the cells and solution by dropping between the culture vessels.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a US National Stage of International Application No. PCT/JP2014/072975, filed Sep. 2, 2014, which claims the benefit of Japanese Patent Application No. 2013-189715, filed Sep. 12, 2013, the entire contents of each of which applications are hereby incorporated by reference. 
       TECHNICAL FIELD 
       [0002]    The present invention relates to a culture system and a culture method that perform cell culture using dropping of a solution. 
       BACKGROUND ART 
       [0003]    Adherent cells such as somatic cells adhere to the bottom surface of a culture vessel and form a foothold before repeating cell division and cell elongation to increase the number thereof In this regard, if the increase in the cell number keeps going on, the cells start to scramble for adhesion area on the bottom surface of the vessel since the cells remain adhered to the bottom surface of the culture vessel. Therefore, if the increase in the cell number continues, there will be no space between the cells and eventually the cells will be multi-layered, due to which the cells will suffocate without sufficient nutrients (confluent condition) and result in dead cells. 
         [0004]    Accordingly, when adherent cells are cultured in a culture vessel, culture needs to be performed while detaching the cells from the culture vessel and transferring them to other vessel at a given cell number (given density) before reaching the confluent condition so as to keep sufficient nutrients to go throughout the adherent cells (subculture). 
         [0005]    In order to detach the cells from the culture vessel and transfer them into other vessel, there is a need to use an enzyme solution such as trypsin to degrade the cell adhesion protein to allow the cells to float or use a scraper to physically detach the cells from the culture vessel. In the field of cell culture, it is well known that these methods are unfavorable treatments since they will place stress on the cells. For example, trypsin is known to be toxic to cells and it is known that a trypsin treatment will change the property of the cells and result dead cells. Therefore, it is desirable to minimize such treatments as much as possible that will place stress on cells. 
         [0006]    In addition, the operation of detaching adherent cells from a culture vessel and transferring them into other vessel is manually conducted in a clean bench. Manual operations are constantly at a risk of contamination. 
       SUMMARY OF THE INVENTION 
       [0007]    Furthermore, in a case of a large volume culture solution (several hundreds ml), there is a need of performing efficient cell culture in an automatic manner without manually transferring the solution. 
         [0008]    Accordingly, in order to subculture adherent cells, the adherent cells need to be moved to another position so as to provide the cells with new environment where they can absorb nutrients, for example, by transferring them into other vessel or the like by manual operation that is associated with a risk of contamination and that places stress on the adherent cells. Thus, automation has been difficult. 
         [0009]    An objective of the present invention is to provide a culture system and method which are capable of automating cell culture by dispersing cells such as adherent cells in a solution and controlling the transfer of the cell-dispersed solution. 
         [0010]    In order to solve the above-described problem, the present inventor has gone through keen study, as a result of which succeeded in automatically performing seeding through collection of the cells with less stress on the cells in a sterile state, in the a case of culturing adherent cells, by using an adherent cell mass or by attaching adherent cells onto magnetic particles or a carrier that adsorbs to the particles to allow the cells to disperse in a solution and transferring the solution into the culture vessel or transferring the solution from the culture vessel to outside by height-difference transfer, thereby accomplishing the present invention. 
         [0011]    Thus, the present invention is as follows. 
         [0012]    (1) A culture system including a plurality of housing vessels for accommodating cells and a solution; conduits for connecting the plurality of housing vessels; an opening-closing mechanism for opening and closing the conduits; and a height-difference imparting mechanism for imparting a height difference between the plurality of housing vessels in order to allow dropping of the cells and the solution between the plurality of housing vessels. 
         [0013]    (2) The culture system according to (1), comprising a first holding section for holding the plurality of housing vessels. (3) The culture system according to (1), wherein the height-difference imparting mechanism imparts a height difference to at least one of the plurality of housing vessels by moving the first holding section. (4) The culture system according to any one of (1) to (3), wherein the plurality of housing vessels comprise one or a plurality of culture vessels. (5) The culture system according to any one of (1) to (4), wherein at least one of the plurality of housing vessels is connected to at least one cylinder or bellows vessel. (6) The culture system according to any one of (1) to (5), wherein the height-difference imparting mechanism imparts a height difference between the housing vessel and the cylinder or the bellows vessel so as to transfer the cells and the solution between the housing vessel and the cylinder or the bellows vessel by dropping. 
         [0014]    (7) The culture system according to (6), wherein the cylinder or the bellows vessel is a waste fluid vessel, and the conduit connecting between the waste fluid vessel and the housing vessel is provided with a cell capturing unit. (8) The culture system according to (7), wherein the cylinder or the bellows vessel is a feed fluid vessel, which is connected to the cell capturing unit. (9) The culture system according to (8), comprising a switching mechanism for switching between the connection between the cell capturing unit and the waste fluid vessel and the connection between the cell capturing unit and the feed fluid vessel. (10) The culture system according to any one of (5) to (9), comprising a stretching mechanism for stretching the cylinder or the bellows vessel. 
         [0015]    (11) The culture system according to any one of (5) to (10), comprising a first holding section for holding at least one of the housing vessels and a second holding section for holding the cylinder or the bellows vessel. (12) The culture system according to (11), wherein the first holding section and the second holding section are detachable. (13) The culture system according to any one of (1) to (12), wherein the housing vessels are provided with a temperature regulating unit. (14) The culture system according to any one of (1) to (13), wherein the housing vessels are provided with a carbon dioxide supply section. (15) The culture system according to any one of (1) to (14), wherein the housing vessels are provided with a ventilation section. (16) The culture system according to any one of (1) to (15), wherein the housing vessels are disposable. 
         [0016]    (17) The culture system according to any one of (5) to (12), wherein the bellows vessel is disposable. (18) The culture system according to any one of (1) to (17), comprising a swing mechanism for swinging the housing vessels. 
         [0017]    (19) The culture system according to any one of (1) to (18), comprising magnetic particles that are attached to the cells in the solution, a magnet provided outside the culture vessel and a magnetic force regulating unit for regulating the magnetic force of the magnet, wherein the magnetic force regulating unit regulates the magnetic force of the magnet to shake or vibrate the magnetic particles and the cells in the culture vessel. (20) The culture system according to any one of (1) to (19), comprising a controller for controlling the opening-closing mechanism and the height-difference imparting mechanism, wherein the controller controls the opening-closing mechanism and the height-difference imparting mechanism according to a predetermined procedure to perform cell culture and transfer the solution in an automatic manner. 
         [0018]    (21) A method for performing cell culture by using the culture system according to any one of (1) to (20). (22) The method according to (21), comprising the steps of: culturing at a position where the height difference between the plurality of housing vessels is made smaller by the height-difference imparting mechanism; transferring the cells and the solution at a dropping position where the height difference between the plurality of housing vessels is made larger by the height-difference imparting mechanism; and treating the solution using at least one cylinder or bellows vessel connected to at least one of the plurality of housing vessels. (23) The method according to (22), wherein the step of treating the solution is a step of discarding the solution or a step of supplying the solution. 
         [0019]    According to the present invention, cell culture can be performed while automatically controlling the position of the cells such that the cells can absorb nutrients, without placing stress on the cells. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0020]      FIG. 1  is a plan view showing a culture system according to an embodiment of the present invention. 
           [0021]      FIG. 2  is a side view showing a variation that can be applied to the culture system shown in  FIG. 1 . 
           [0022]      FIG. 3  is a plan view showing the variation of  FIG. 2 . 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0023]    A culture system and a culture method according to an embodiment of the present invention will be described with reference to the drawings. Cells that are cultured by the culture system and the culture method of the present invention are not limited to adherent cells and they may be floating cells. In a case of culturing adherent cells, a adherent cell mass can be used to allow dispersion of the adherent cells in a solution, or adherent cells can be attached to magnetic particles or a carrier that adsorbs to the magnetic particles (fiber assembly or a porous solid material) to allow dispersion of the adherent cells in a solution. Accordingly, the adherent cells can move together with the solution, and culture can automatically be controlled using height-difference transfer. 
         [0024]    A culture system  1000  according to the embodiment of the present invention will be described with reference to  FIG. 1 . The culture system  1000  is provided with a main stage  100  (first holding section) that performs cell culture, a height-difference imparting mechanism  200  that tilts the stage  100 , and a substage  300  (second holding section) that can be connected with the main stage  100 . The main stage  100  can be moved between a horizontal state and a vertical state by the height-difference imparting mechanism  200 . 
         [0025]    The main stage  100  is provided with a preparatory culture vessel  110  having a flat resin culture space, and a main culture vessel  120  having a flat resin culture space. The volume of the preparatory culture vessel  110  is smaller than the volume of the main culture vessel  120 . 
         [0026]    For example, the volume of the preparatory culture vessel  110  may be 10-30 ml or the volume of the main culture vessel  120  may be 100-300 ml. Each of the culture vessels may be disposable. Moreover, although the shape of each culture vessel is a flat cuboid, it is not limited thereto and any shape such as a column or a shape having a bottom surface of a column with a circular cone on it. Furthermore, the inner surface of each culture vessel is preferably treated such that adherent cells do not adhere onto it. 
         [0027]    The preparatory culture vessel  110  is provided with a gas supply section  111  for supplying CO 2  gas inside the vessel, a ventilation filter  112  for appropriately managing the pressure inside the vessel, a temperature regulating unit  113  for regulating the temperature of the solution inside the vessel, and a feed port  114  for introducing a solution, cells or the like into the vessel. The gas supply section  111  and the ventilation filter  112  are connected to the preparatory culture vessel  110  via a flexible resin connecting tube (conduit). In addition, the preparatory culture vessel  110  and the main culture vessel  120  are also connected with a flexible resin connecting tube. The connecting tubes are provided with valves  171  or  172 , respectively. The valve  173  provided on the tube between the preparatory culture vessel  110  and the main culture vessel  120  may be a check valve. The feed port  114  is closed with a cap. 
         [0028]    The main culture vessel  120  is provided with a gas supply section  121  for supplying CO 2  gas inside the vessel, a ventilation filter  122  for appropriately managing the pressure inside the vessel, a temperature regulating unit  123  for regulating the temperature of the solution inside the vessel to a suitable temperature, and a feed port  124  for introducing a solution, cells or the like into the vessel. The gas supply section  121  and the main culture vessel  120  are connected via a flexible resin connecting tube having a valve  174 . The ventilation filter  122  and the main culture vessel  120  are connected via a flexible resin connecting tube equipped with a valve  175 . The feed port  124  is closed with a cap. 
         [0029]    The gas supply sections  111  and  121  supply gas having a carbon dioxide concentration and a humidity that are required for cell culture from a CO 2  gas tank (not shown) into the culture vessel. Preferably, gas conditions with a carbon dioxide concentration of 5%, a humidity of 95%, and a temperature of 37° C. can be employed for the cell culture. The temperature regulating units  113  and  123  are preferably, but not limited to, a temperature managing device such as a thermal cycler, a film heater (sheet heating element) or a coolable and heatable Peltier element. The temperature regulating units  113  and  123  may be provided on both top and bottom surfaces or on the top surface of the preparatory culture vessel  110  or the main culture vessel  120 , respectively. 
         [0030]    The main culture vessel  120  is connected to a sorting vessel  130  for sorting the cells cultured in the main culture vessel  120  and to a cell capturing unit  150 . The sorting vessel  130  is a bottle-shaped vessel. The sorting vessel  130  is connected to a ventilation filter  132  for suitably managing the pressure inside the vessel. A connecting tube between the sorting vessel  130  and the ventilation filter  132  is provided with a valve  177 . The cell capturing unit  150  is provided with a filter block  151 , which captures the cells in the solution sent from the main culture vessel  120 . The solution removed of the cells is sent to the substage  300 . 
         [0031]    In the main stage  100 , a plurality of recesses are formed, which accommodate and hold the preparatory culture vessel  110 , the main culture vessel  120 , the sorting vessel  130 , the cell capturing unit  150 , the connecting tubes and the valves  171 - 179 , respectively. As the valves  171 - 179 , solenoid valves that can automatically be opened or closed with a controller or pinchcocks that can manually or automatically be opened or closed can be used. 
         [0032]    The height-difference imparting mechanism  200  is provided with a rotation shaft  210  that is integrated with and thus rotates with the main stage  100 , a motor  220  and a transmitting mechanism  230  for transmitting the rotation of the motor to the rotation shaft. The transmitting mechanism may be a belt, a gear or the like. 
         [0033]    The substage  300  is provided with a feed fluid bellows vessel  310  and a waste fluid bellows vessel  320 . The feed fluid bellows vessel  310  is provided with a feed port  314  for introducing a solution into the vessel  310 . The feed fluid bellows vessel  310  and the waste fluid bellows vessel  320  are connected to stretching mechanisms  311  and  321  for stretching the respective bellows vessels, respectively. The feed fluid bellows vessel  310  and the waste fluid bellows vessel  320  may be made from a flexible resin and may be disposable. The feed fluid bellows vessel  310  and the waste fluid bellows vessel  320  are connected to the cell capturing unit  150  via a connecting tube equipped with a three-way selector valve (switching mechanism)  301 . The three-way selector valve  301  switches between the connection between the cell capturing unit  150  and the feed fluid bellows vessel  310  and the connection between the cell capturing unit  150  and the waste fluid bellows vessel  320 . The connection state of the three-way selector valve  301  can be switched by the controller. The feed port  314  is closed with a cap. 
         [0034]    The substage  300  also has a plurality of recesses formed, which accommodate and hold the feed fluid bellows vessel  310 , the stretching mechanism  311 , the waste fluid bellows vessel  320 , the stretching mechanism  321 , the connecting tubes and the three-way selector valve  301 , respectively. 
         [0035]    The main stage  100  is provided with a plurality of arm parts  170  that stretch by the controller while the substage  300  is provided with a plurality of arm joints  330  for receiving the plurality of arm parts  170 . Once the arm parts  170  elongate and join the arm joints  330 , the subunit  300  integrates with the main unit  100  so that the subunit  300  can be rotated together with the main unit. 
         [0036]    Hereinafter, an operation of the cell culture system  1000  of the present embodiment will be described. 
         [0037]    (Preparatory Culture) 
         [0038]    First, the preparatory culture step carried out in the preparatory culture vessel  110  will be described. The main stage  100  and the substage  300  are separately arranged at substantially horizontal culture positions. At these culture positions, cells and a solution are injected from the feed port  114  into the preparatory culture vessel  110  using a dispensing mechanism while CO 2  gas at a suitable concentration is supplied from the gas supply section  111 . In a state where the cells, the culture solution and the CO 2  gas are supplied into the preparatory culture vessel  110 , the temperature regulating unit  113  controls the temperature inside the preparatory culture vessel  110  to have a suitable temperature while the motor  220  rotates the rotation shaft  200  at a predetermined angle range to periodically tilt (shake, swing) the main stage  100  as a whole. Due to this tilting, the cells, the culture solution and CO 2  in the preparatory culture vessel  110  on the main stage  100  are agitated and undergo culture. 
         [0039]    After a predetermined period of time following the initiation of culture where the cells in the preparatory culture vessel  110  have sufficiently been proliferated, the height-difference imparting mechanism  200  rotates the main stage  100  and halts the preparatory culture vessel  110  at a height-difference transfer position. The height-difference transfer position is preferably such that the main stage  100  is kept vertical, but it is not limited thereto as long as there is a tilt that allows the liquid in the preparatory culture vessel  110  to drop by gravity via the connecting tube. 
         [0040]    When the valves  172 ,  173  and  175  are opened at the height-difference transfer position, the solution containing the cells in the preparatory culture vessel  110  is automatically transferred into the main culture vessel  120  by gravity. The valve  172  may be closed and the valve  171  may be opened so that gas is injected from the gas supply section  111  into the preparatory culture vessel  110 , which promotes transfer of the solution. At the height-difference transfer position, the valves  176  and  179  are closed. 
         [0041]    (Main Culture) 
         [0042]    Next, the main culture step carried out in the main culture vessel  120  will be described. Once the solution is completely transferred from the preparatory culture vessel  110  into the main culture vessel  120 , the valve  173  is closed, and the main stage  100  is moved to the horizontal culture position. At this culture position, the culture solution is injected from the feed port  124  into the main culture vessel  120  using the dispensing mechanism while CO 2  gas at a suitable concentration is supplied from the gas supply section  121 . In a state where the cells, the culture solution and the CO 2  gas are supplied into the main culture vessel  120 , the temperature regulating unit  123  controls the temperature inside the main culture vessel  120  to have a suitable temperature while the motor  220  rotates the rotation shaft  200  at a predetermined angle range to periodically tilt the main stage  100  as a whole. Due to this tilting, the cells, the culture solution and CO 2  in the preparatory culture vessel  120  on the main stage  100  are agitated and undergo culture. 
         [0043]    (Exchanging Culture Solution) 
         [0044]    Continuously, a step of exchanging the culture solution in a case where the culture solution in the main culture vessel  120  is required after a predetermined period of time following initiation of the culture will be described. First, at the culture positions, the arm parts  170  and the arm joints  330  are joined to integrate the main stage  100  and the substage  300 . The height-difference imparting mechanism  200  rotates the main stage  100  and the substage  300  such that the main culture vessel  120  is moved upward with respect to the substage  300  and halted at the height-difference transfer position. 
         [0045]    At the height-difference transfer position, the valve  176  is kept close while the valves  175  and  179  are opened so as to connect the cell capturing unit  150  and the waste fluid bellows vessel  320  with the three-way selector valve  310  on the substage  300 . The solution containing the cells in the main culture vessel  123  is transferred into the cell capturing unit  150  by gravity. In the cell capturing unit  150 , the cells are captured on the top surface of the filter block  151  (surface on the valve  179  side), and the solution removed of the cells drops by gravity and is transferred into the contracted waste fluid bellows vessel  320  to be discarded. Since the waste fluid bellows vessel  320  is contracted in advance as shown in  FIG. 1 , the transfer of the solution from the main culture vessel  123  via the cell capturing unit  150  can be promoted as the waste fluid bellows vessel  320  is elongated using the stretching mechanism  321 . 
         [0046]    Once the solution is completely discarded, the three-way selector valve  301  separates the waste fluid bellows vessel  320  and the cell capturing unit  150  while the height-difference imparting mechanism  200  moves the main stage  100  and the substage  300  to the fluid feeding positions. At the fluid feeding position, the main stage  100  and the substage  300  are generally vertical while the feed fluid bellows vessel  310  is positioned above the main culture vessel  120 . The main stage  100  and the substage  300  are not necessarily vertical as long as the main culture vessel  120  side of the main stage  100  is tilted downward while the feed fluid bellows vessel  310  of the main state  100  and the substage  300  is tilted upward. As the main stage  100  and the substage  300  move from the height-difference transfer positions to the fluid feeding positions, the filter block  151  of the cell capturing unit  150  turns upside down. By this, the cells captured on the top surface of the filter block  151  at the height-difference transfer position will stay on the bottom surface of the filter block  151  at the fluid feeding position. 
         [0047]    At the fluid feeding position, the three-way selector valve  301  connects the feed fluid bellows vessel  310  and the cell capturing unit  150  while the valves  179  and  175  are opened. In this state, a new culture solution is transferred by gravity from the feed fluid bellows vessel  310  that has already been injected with the new culture solution beforehand into the cell capturing unit  150 . The culture solution is transferred into the main culture vessel  120  together with the cells captured on the bottom surface (valve  179  side) of the filter block  151  of the cell capturing unit  150 , thereby completing fluid feeding. Accordingly, the height-difference imparting mechanism  200  is used to turn the cell capturing unit  150  upside down so that the cells can be separated from the culture solution and the cells can be dispersed into the culture solution with the cell capturing unit  150  in an automatic manner. Here, by compressing the feed fluid bellows vessel  310  with the stretching mechanism  311 , transfer of the new culture solution can be promoted. 
         [0048]    Once the fluid feeding is completed, the height-difference imparting mechanism  200  moves the main stage  100  and the substage  300  to the culture positions to repeat cell culture. 
         [0049]    (Cell Sorting) 
         [0050]    Finally, the cell sorting step will be described. After a predetermined period of time following the initiation of culture and the cells in the main culture vessel  120  have sufficiently been proliferated, the height-difference imparting mechanism  200  rotates the main stage  100  and halts the main culture vessel  120  at the height-difference transfer position. At the height-difference transfer position, the valves  175 ,  176  and  177  are opened while the valve  179  is closed and thus the solution containing the cells drops by gravity from the main culture vessel  120  into the sorting vessel  130 , where the cells are sorted. Furthermore, by closing the valve  175  and opening the valve  174  to inject gas from the gas supply section  121  into the main culture vessel  120 , the transfer of the solution from the main culture vessel  120  into the sorting vessel  130  can be promoted. 
         [0051]    (Variation) 
         [0052]    A variation of the present embodiment will be described with reference to  FIGS. 2 and 3 . This variation is provided with a sliding mechanism  414  for sliding the preparatory culture vessel  110  and/or the main culture vessel  120 , and a magnetic force regulating unit (shaking mechanism)  407  for shaking the solution in the preparatory culture vessel  110  and/or the main culture vessel  120  by magnetic force. 
         [0053]    The culture vessel  110  ( 120 ) is connected to the sliding mechanism  414  which periodically slides (vibrates) the culture vessel in the horizontal direction as indicated by the arrow  414   a  shown in the figure. The sliding mechanism  414  can be realized by converting the rotation of the motor into linear motion with a rack or a cam. Since the position of the culture vessel  110  ( 120 ) alters by the sliding mechanism  414 , the connecting tube connected to the culture vessel  110  ( 120 ) is arranged to have enough length to maintain the connection even when the position of the culture vessel  110  ( 120 ) changes. 
         [0054]    The magnetic force regulating unit  407  can get closer to or away from the culture vessel  110  ( 120 ) along a guide  407   b  with a magnetic force regulating unit moving mechanism  407   c.  When the magnetic force regulating unit  407  gets closer to the bottom surface of the culture vessel  110  ( 120 ), the magnetic force of the magnets  407   a  can concentrate and fix (adsorb) the magnetic particles and the cells contained in the culture solution in the culture vessel  110  ( 120 ) on the inner bottom surface of the culture vessel  110  ( 120 ). When the magnetic force regulating unit  407  gets away from the bottom surface of the culture vessel  110  ( 120 ), the magnetic force of the magnets  407   a  no longer has the effect inside the culture vessel  110  ( 120 ), and thus the magnetic particles and the cells move away from the inner bottom surface of the culture vessel  110  ( 120 ) and disperse. 
         [0055]    As shown in  FIG. 3 , a plurality of permanent magnets  407   a  are arranged in a matrix, i.e., vertically and horizontally arrayed at regular intervals, in the magnetic force regulating unit  407 . Each of the permanent magnets  407   a  included in this array can concentrate and fix the magnetic particles and the cells contained in the culture solution in the culture vessel  110  ( 120 ) onto the inner surface of the culture vessel  110  ( 120 ). The polarities of the adjacent magnets  407   a  are always opposite. Accordingly, polarities of magnetic particles that adsorbed onto adjacent magnets  407   a  differ from each other and result repulsive force between them, as a result of which the populations of the magnetic particles and the cells adsorbed onto the magnets  407   a  are more likely to concentrate. Here, by providing a magnet for adsorbing magnetic particles on the sorting vessel  130 , the magnetic particles can be separated from the cells. 
         [0056]    Since conventional culture systems use a cylinder or a pump, they require washing of the cylinder and are also associated with a risk of contamination. The culture vessel, the sorting vessel and the bellows vessel of the culture system of the present embodiment can all essentially be made from plastic and thus can be completely disposable, which can significantly reduce the risk of contamination. Although a risk of contamination increases, a cylinder can be used instead of each bellows vessel. While the solution movement between the respective culture vessels or between the culture vessel and the bellows vessel was realized through dropping using height difference, the solution movement can be promoted by suction with a vacuum pump. Although two culture vessels are used in the present embodiment, the number of the culture vessels is not limited thereto and may be one or three. The controller of the present embodiment is connected to an operating panel, where various settings of the program can be changed to perform culture.