Abstract:
Disclosed is a rotating culture vessel based on a rotating culture technology using an RWV, by which cell seeding, liquid medium exchange, quality control and so on can be automated and degassing can be conducted simultaneously with liquid medium exchange without disturbing the cells under culture. Also disclosed is an automatic cell culture apparatus using the same. A rotating culture vessel, which contains cells and a liquid culture medium, to be attached to a horizontal rotating shaft of a rotating culture device to three-dimensionally culture the cells, wherein one or more inlets/outlets for supplying cells and a liquid culture medium at the early stage and then taking out the cultured cells, are formed at appropriate position of a flat cylindrical culture container; at least one pair of a supply port and a discharge port for liquid medium exchange is provided on the outer circumferential cylindrical face of the culture container.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a rotating culture vessel and an automatic cell culture apparatus using the vessel, and more specifically relates to a rotating culture vessel suited for automatic exchange of a liquid culture medium and an automatic cell culture apparatus having an automatic liquid culture medium exchange function. 
       BACKGROUND ART 
       [0002]    Conventionally, various automatic cell culture apparatuses have been supplied. For example, Patent Document 1 pertaining to a prior application of the present applicants discloses an automatic cell culture system. The automatic cell culture system can handle a plurality of culture cassettes containing a culture dish, automatically transfer the culture cassette with a robot arm for a dispensing operation and the like, control atmospheres such as gas concentration, temperature, and humidity in an incubator, and reliably prevent not only contamination in the incubator but also contamination during transfer of the culture dish for a dispensing operation and the like. 
         [0003]    Patent Documents 2 and 3 also disclose an automatic cell culture apparatus that automates, for example, an exchange operation of a liquid culture medium with a general purpose multiple joints type robot in a two-dimensional culture apparatus. The automatic cell culture apparatus has an effect on the prevention of bacterial invasion by minimization of human intervention, but an expensive general purpose industrial robot performs the same operations as those that a human has performed, and such an apparatus requires high cost. Moreover, such an apparatus is not beyond common two-dimensional cell culture apparatuses and is not an apparatus for achieving three-dimensional culture. 
         [0004]    Meanwhile, mesenchymal stem cells derived from the bone marrow has multipotency to be differentiated into tissues such as bone, cartilage, adipose, and ligament. However, it is known that, in a common in vitro culture, the cells are settled to the bottom of a petri dish due to the Earth&#39;s gravity to form a two-dimensional sheet and the original cell characteristics disappear. To address this, a culture method using a rotating wall vessel (RWV) bioreactor has been developed in order to achieve the three-dimensional culture in a microgravity environment that is nearly gravity-free, and cells are three-dimensionally cultured with appropriate differentiation inducing factors to be differentiated into an original tissue of the cell. A culture apparatus using the RWV is released from Synthecon. The apparatus is composed of a flat cylindrical vessel having a gas permeable membrane on the back side, and the vessel is rotated around an attaching part to which a horizontal rotating shaft of a rotation controller is attached. The rotation of the vessel constantly changes the gravity direction to cells, and consequently can generate a microgravity environment having one-hundredth the ground gravity by time average. Hence, clumps of cells are not settled and can be cultured while floating. The apparatus is typically referred to as a rotating culture device. 
         [0005]    The RWV used in the conventional rotating culture device includes an inlet/outlet for a cell suspension and a pair of a supply port and a discharge port for a liquid culture medium on the front face orthogonal to the rotating shaft, and the liquid culture medium is exchanged manually. That is, for liquid culture medium exchange, the RWV is removed from the rotating shaft and left with the supply port and the discharge port facing upward, a rubber cap of the supply port is taken off, a leading end of a supply syringe including a new liquid culture medium is closely connected to the supply port, while a leading end of an empty discharge syringe is closely connected to the discharge port, then cocks of the supply port and the discharge port are unlocked, a piston of the supply syringe is pushed by one hand to supply the liquid culture medium into the vessel, and simultaneously a piston of the discharge syringe is pulled by the other hand to suck the used liquid culture medium in the vessel. The culture period of cells is typically about two weeks, and a culture medium is required to be exchanged every several days. Thus, the culture medium exchange operation that is complicated and requires a lot of skill must be carried out in each case. Such a manual culture medium exchange operation requires a lot of labor and time. When many RWVs are run, the exchange operation becomes a heavy burden as well as unexpected environmental contamination may occur during the culture medium exchange operation. In addition, the vessel having the cell suspension inlet, the supply port, and the discharge port on the front face has an disadvantage of small view for the observation of cells in the vessel. 
         [0006]    To address this, the present applicant has developed an automatic cell culture apparatus as disclosed in Patent Document 4. Namely, the automatic cell culture apparatus includes a closed housing and a rotating culture device in the closed housing. The rotating culture device includes a cylindrical culture container having a horizontal rotating shaft, a cell suspension inlet, a supply port, and a discharge port, and each of the supply port and the discharge port has a septum seal structure for liquid culture medium exchange. The supply port and the discharge port are provided as a pair on the outer circumferential cylindrical face of the culture container at positions 180° apart from each other with respect to the rotating center toward the radial direction. The automatic cell culture apparatus also includes a syringe shifting means for inserting/withdrawing an injection needle of a supply syringe containing a new liquid culture medium to/from the supply port and for inserting/withdrawing an injection needle of an empty discharge syringe to/from the discharge port while locating the supply port and the discharge port on a vertical line with the supply port facing upward and includes a piston driving means for pushing a piston of the supply syringe while inserting the supply syringe into the supply port and for simultaneously pulling a piston of the discharge syringe while inserting the discharge syringe into the discharge port. 
         [0007]    The apparatus thereby has a function of automatically exchanging a liquid culture medium in the rotating culture device using the RWV. Hence, such an apparatus can largely reduce burdens of culture operators such as researchers and achieve efficient cell culture as well as minimize the possibility of contamination in culture. When a plurality pairs of the supply ports and the discharge ports are provided on the outer circumferential cylindrical face of the culture container at an equal angle interval depending on a culture period and the number of liquid culture medium exchanges, the supply port and the discharge port can be newly used for every liquid culture medium exchange. Thus, such an apparatus has an advantage of minimizing the contamination during the liquid culture medium exchange. 
       Citation List 
       [0008]    Patent Literature 
         [0009]    Patent Document 1: JP-A No. 2006-014675 
         [0010]    Patent Document 2: JP - A No. 2006-115798 
         [0011]    Patent Document 3: JP-A No. 2006-149268 
         [0012]    Patent Document 4: JP - A No. 2008-237203 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0013]    However, the automatic cell culture apparatus disclosed in Patent Document 4 still has some problems. That is, air may not be completely substituted at the initial charging of a cell suspension to remain in the vessel, or air may be introduced into the vessel in culture through a permeable membrane on the backside. Accumulated gas in the vessel disturbs the flow of a liquid culture medium to provide irregular load to cells during rotation culturing, and hence air bleeding is essential. In conventional exchange of a liquid culture medium, a vessel is positioned with the supply port and the discharge port for a liquid culture medium in the vertical direction and with the supply port locating at the upper position, and a liquid culture medium is supplied with the supply syringe from the top and is sucked with the discharge syringe from the bottom. Hence, gas accumulated in the upper part of the vessel cannot be bled. 
         [0014]    Therefore, in view of the above circumstances, it is an object of the present invention to provide a rotating culture vessel that can automate cell seeding, liquid culture medium exchange, quality control, and the like based on the rotation culture technique using the RWV and that can simultaneously bleed air during the liquid culture medium exchange without disturbing cultured cells and an automatic cell culture apparatus using the vessel. 
       Solution to Problem 
       [0015]    In order to solve the above problems, the present invention provides a rotating culture vessel used for three-dimensional culture of a cell in a nearly gravity-free microgravity environment, the rotating culture vessel including a cell and a liquid culture medium and being attached to a horizontal rotating shaft of a rotating culture device. The rotating culture vessel is characterized by including one or more inlets/outlets for supplying a cell and a liquid culture medium at a initial stage and taking out a cultured cell at an appropriate position on a flat cylindrical culture container, and at least one pair of a supply port and a discharge port for liquid culture medium exchange on an outer circumferential cylindrical face of the culture container. The pair of the supply port and the discharge port are positioned 180° opposite to each other, the discharge port has a center line passing through a rotating center, and the supply port has a center line eccentrically positioned with respect to the rotating center (claim  1 ). 
         [0016]    It is more preferable that a distance between the center line of the supply port and the rotating center is 0.5r to 0.9r where the culture container has a culture space having a radius of r (claim  2 ). 
         [0017]    It is also preferable that three pairs of the supply ports and the discharge ports are provided on the outer circumferential cylindrical face of the culture container and the supply ports and the discharge ports are provided at an equal angle interval (claim  3 ). 
         [0018]    It is preferable that each of the supply port and the discharge port has a septum seal structure, a supply syringe used for liquid culture medium exchange has a leading end having an injection needle optionally passing through the septum seal, a discharge syringe used for liquid culture medium exchange has a leading end having an injection needle optionally passing through the septum seal, a new liquid culture medium is supplied from the supply syringe while a used liquid culture medium is sucked with the discharge syringe, and air is optionally bled with the discharge syringe (claim  4 ). 
         [0019]    The outer circumferential cylindrical face of the culture container includes a cell suspension supply port for supplying a cell suspension composed of a cell and a liquid culture medium, a front face orthogonal to the rotating shaft of the culture container includes an air bleeding port and a cell discharge port, each of the cell suspension supply port and the air bleeding port has a septum seal structure, and the cell discharge port has a rubber plug structure with a large opening (claim  5 ). 
         [0020]    Each port having the septum seal structure includes an inlet flow path having a large diameter for accepting the injection needle behind the septum seal and an orifice flow path having a small diameter between the inlet flow path and the culture space of the culture container (claim  6 ). 
         [0021]    The present invention also provides an automatic cell culture apparatus using the rotating culture vessel and used for three-dimensional culture of a cell in a nearly gravity-free microgravity environment. The automatic cell culture apparatus is characterized by including a closed housing having an air conditioning function, an incubator box of a rotating culture device in a middle chamber of the closed housing, a cool supply box storing a supply syringe for supplying a liquid culture medium in a lower chamber of the closed housing, and a cool discharge box storing a discharge syringe for collecting a liquid culture medium in an upper chamber of the closed housing. Each of the incubator box, the cool supply box, and the cool discharge box includes a front face having an automatically operated door. The rotating culture device has a shaft direction shifting means shifting a horizontal rotating shaft back and forth in a shaft direction, the horizontal rotating shaft is provided in the incubator box, and the rotating shaft has an end removably installed with the rotating culture vessel. A front space of the cool supply box includes an XYZ-axis shifting mechanism for supply, the XYZ-axis shifting mechanism for supply includes a movable part for supply driven by the mechanism, and the movable part for supply has a fixing chuck for holding the supply syringe upward and a push up means for pushing a piston up. A front space of the cool discharge box includes an XYZ-axis shifting mechanism for discharge, the XYZ-axis shifting mechanism for discharge includes a movable part for discharge driven by the mechanism, and the movable part for discharge has a fixing chuck for holding the discharge syringe downward and a pull up means for pulling a piston up. Each door is opened, the shaft direction shifting means is driven to bring the rotating culture vessel from the incubator box to a front space, and the rotating culture vessel is stopped with the supply port facing downward and the discharge port facing upward. The XYZ-axis shifting mechanism for supply and the fixing chuck are driven to take out the supply syringe from the cool supply box and to locate the supply syringe below the rotating culture vessel, and the XYZ-axis shifting mechanism for discharge and the fixing chuck are driven to take out the discharge syringe from the cool discharge box and to locate the discharge syringe above the rotating culture vessel. The XYZ-axis shifting mechanism for supply is driven to airtightly connect the supply syringe to the supply port, simultaneously the XYZ-axis shifting mechanism for discharge is driven to airtightly connect the discharge syringe to the discharge port, and then the push up means and the pull up means are synchronously driven to exchange a liquid culture medium in the rotating culture vessel (claim  7 ). 
         [0022]    Each of the cool supply box and the cool discharge box includes a rotating revolver type stacker and a plurality of holders around the stacker. The stacker has a vertical rotating shaft controlled with a stepping motor, and each holder elastically removably holds the syringe in a transverse direction with the syringe in the vertical direction (claim  8 ). 
         [0023]    It is preferable that the rotating culture device includes a plurality of the rotating shafts shifted back and forth by a common shaft direction shifting means, the rotating shafts are not overlapped in the vertical direction, ends of the rotating shafts are displaced back and forth, and the adjacent rotating culture vessels do not interfere with each other in the case of the rotating culture vessels being installed to the corresponding rotating shafts (claim  9 ). 
       Advantageous Effects of Invention 
       [0024]    The rotating culture vessel of the present invention as described above includes one or more inlets/outlets for supplying cells and a liquid culture medium at a initial stage and taking out cultured cells at an appropriate position on a flat cylindrical culture container and at least one pair of a supply port and a discharge port for liquid culture medium exchange on an outer circumferential cylindrical face of the culture container. The pair of the supply port and the discharge port are positioned 180° opposite to each other, the discharge port has a center line passing through a rotating center, and the supply port has a center line eccentrically positioned with respect to the rotating center. Hence, the rotating culture vessel is stopped with the supply port facing downward and the discharge port facing upward, the supply syringe including a new liquid culture medium is connected to the supply port, while the discharge syringe is connected to the discharge port, the piston of the supply syringe is pushed up to supply the new liquid culture medium into the culture container, and the piston of the discharge syringe is simultaneously pulled up to suck a used liquid culture medium for liquid culture medium exchange, as well as at the time, gas accumulated in the upper part of the culture container can be sucked with the discharge syringe. The center line of the supply port is eccentrically positioned with respect to the rotating center, hence, even when clumps of cells in culture are settled down to be placed in the lower part of the culture container, disassembling of the clumps of cells due to the flow caused by the supply of the liquid culture medium can be suppressed because the supply port is placed avoiding the clumps of cells. In particular, the effect is increased when the distance between the center line of the supply port and the rotating center is 0.5r to 0.9r where the culture container has a culture space having a radius of r. The culture container does not have a supply port and a discharge port interrupting the view on the front face, and thus the progress of culture is easily observed. 
         [0025]    Three pairs of the supply ports and the discharge ports are provided on the outer circumferential cylindrical face of the culture container, and thus a liquid culture medium can be exchanged three times. Hence, the culturing can be continued for about two weeks that is a common cell culture period, and the supply port and the discharge port can be newly used for every exchange of a liquid culture medium. Therefore, the occurrence of contamination can be minimized. When the supply ports and the discharge ports are provided at an equal angle interval, the supply port and the discharge port can be readily located at exact positions. 
         [0026]    When each of the supply port and the discharge port has a septum seal structure and each of the supply syringe and the discharge syringe used for liquid culture medium exchange has a leading end with an injection needle that can pass through the septum seal, the passing of the injection needle through the septum seal can achieve airtight connection, and a new liquid culture medium is supplied from the supply syringe while a used liquid culture medium is sucked with the discharge syringe as well as air can be bled with the discharge syringe. Furthermore, when the injection needle is removed from the septum seal, the hole formed with the injection needle is self-repaired to be closed. Thus, the supply port and the discharge port do not require a special structure for switching operation, and the supply port and the discharge port can be downsized. Therefore, the outer circumferential cylindrical face of the culture container can have three pairs of the supply ports and the discharge ports. 
         [0027]    The outer circumferential cylindrical face of the culture container includes the cell suspension supply port for supplying a cell suspension composed of cells and a liquid culture medium, and a front face orthogonal to the rotating shaft of the culture container includes an air bleeding port and a cell discharge port. Hence, the culture container is kept in a horizontal position with the front face facing upward, a cell suspension supply syringe is connected to the cell suspension supply port, an air discharge syringe is connected to the air bleeding port, a cell suspension is supplied from the cell suspension supply syringe, air in the culture container is simultaneously sucked with the air discharge syringe, the air in the culture container is replaced with the cell suspension, and initial culture preparation can be completed. In particular, when each of the cell suspension supply port and the air bleeding port has the septum seal structure, an insertion of the injection needle of the syringe to the septum seal can simply achieve airtight connection to lead to easy manual operation. The cell discharge port has a rubber plug structure with a large opening, and hence clumps of cells can be readily taken out after culture. 
         [0028]    Each port having the septum seal structure includes the inlet flow path having a large diameter for accepting the injection needle behind the septum seal and the orifice flow path having a small diameter between the inlet flow path and the culture space of the culture container. Thus, even when the injection needle is inserted into the septum seal at a slightly dislocated position, the injection needle can be accepted into the inlet flow path, and the supply syringe can supply a liquid culture medium through the orifice flow path into the culture space. Hence, such a structure can minimize the disturbance in the culture space due to the liquid culture medium flow and can suppress the introduction of air remaining in the inlet flow path through the orifice flow path into the culture space in rotation culture. Furthermore, the circumferential face of the culture space has a small hole of the orifice flow path alone, and thus the liquid culture medium is not disturbed in rotation culture. 
         [0029]    The automatic cell culture apparatus of the present invention is based on the rotation culture technique using RWVs and has the function of automatic liquid culture medium exchange. Thus, such an apparatus can largely reduce burdens of culture operators such as researchers to achieve efficient cell culture as well as can minimize the possibility of contamination in culture. Hence, even medical institutions not having CPC in compliance with GMP can expect clinical application of regenerative medicine, and consequently, the regenerative medicine can be greatly generalized. The present invention can completely automate the liquid culture medium exchange operation that has been proven by conventional manual operation in which a new liquid culture medium is supplied into a culture container in a rotating culture vessel from a supply port with a supply syringe and a used liquid culture medium is simultaneously sucked from a discharge port with a discharge syringe. That is, the new liquid culture medium is supplied into the culture space of the rotating culture vessel from the supply port facing downward with the supply syringe, the used liquid culture medium is simultaneously sucked from the discharge port facing upward with the discharge syringe, and concurrently air can be bled. The center line of the supply port is eccentrically positioned with respect to the rotating center, and thus the disassembling of the clumps of cells can be suppressed as described above. The used liquid culture medium sucked in the discharge syringe is stored in the cool discharge box, and hence the contamination can be checked later. 
         [0030]    Typically, each of the supply port and the discharge port is used only once for the liquid culture medium exchange, but the rotating culture vessel has a comparatively small culture space, for example, a small vessel has a volume of 10 ml and a large vessel has a volume of 20 ml. Thus, the numbers of the supply ports and the discharge ports are limited on the outer circumference. In the case of a long term culturing, each of the supply port and the discharge port may be used twice or more. In such a case, the numbers of the supply syringes and the discharge syringes require more than the number of the supply ports, but the use of a rotating revolver type stacker having a plurality of holders around the stacker can achieve the supply and the storage of the supply syringes and the discharge syringes. 
         [0031]    Even in the case that each of the supply port and the discharge port is used once for the liquid culture medium exchange, when the rotating culture device includes a plurality of rotating shafts, a plurality of rotating culture vessels are installed to the ends, and a plurality of cell lines are simultaneously cultured, it is required that the numbers of the supply syringes and the discharge syringes, obtained by multiplying the number of the vessels by the number of pairs of the supply ports and the discharge ports. In such a case, the rotating revolver type stacker having a plurality of holders around the stacker can be used. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0032]      FIG. 1  is a general perspective view of an automatic cell culture apparatus of the present invention except for a part of an outer cover. 
           [0033]      FIG. 2  is a side view showing the internal structure of the automatic cell culture apparatus of the present invention. 
           [0034]      FIG. 3  is an elevation view showing the internal structure of the automatic cell culture apparatus of the present invention. 
           [0035]      FIG. 4  is a perspective view showing a mechanism part of a rotating culture device having a shaft direction shifting means. 
           [0036]      FIG. 5  is a perspective view showing a XYZ-axis shifting mechanism for supply. 
           [0037]      FIG. 6  is a perspective view showing a XYZ-axis shifting mechanism for discharge. 
           [0038]      FIG. 7  is a perspective view of a cool supply box having a rotating revolver type stacker. 
           [0039]      FIG. 8  is a perspective view of a cool discharge box having the rotating revolver type stacker. 
           [0040]      FIG. 9  is a perspective view of a rotating culture vessel of the present invention viewed from the front side. 
           [0041]      FIG. 10  is a perspective view of the rotating culture vessel of the present invention viewed from the back side. 
           [0042]      FIG. 11  is an elevation view of the rotating culture vessel. 
           [0043]      FIG. 12  is a left side view of the rotating culture vessel. 
           [0044]      FIG. 13  is a back view of the rotating culture vessel. 
           [0045]      FIG. 14  is a cross-sectional view taken along the line A-A in  FIG. 11 . 
           [0046]      FIG. 15  is a cross-sectional view taken along the line B-B in  FIG. 11 . 
           [0047]      FIG. 16  is a schematic diagram showing the liquid culture medium exchange operation using the first pair of the supply port and the discharge port. 
           [0048]      FIG. 17  is a schematic diagram showing the liquid culture medium exchange operation using the second pair of the supply port and the discharge port. 
           [0049]      FIG. 18  is a schematic diagram showing the liquid culture medium exchange operation using the third pair of the supply port and the discharge port. 
           [0050]      FIG. 19  is a photograph in place of a drawing, showing external appearances of cartilage tissues as the results of cartilage tissue formation experiment using bone marrow cells of Japanese white rabbits using the automatic cell culture apparatus of the present invention (automatic culture) and a conventional rotating culture device (manual culture). 
           [0051]      FIG. 20  is a graph showing the result comparing the production amounts of GAG in cartilage matrix. 
           [0052]      FIGS. 21  are micrographs of cartilage tissues stained with Alcian blue. 
           [0053]      FIGS. 22  are micrographs of cartilage tissues stained with toluidine blue. 
           [0054]      FIGS. 23  are micrographs of cartilage tissues stained with HE. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0055]    Next, the present invention will be described in further detail based on embodiments shown in attached drawings.  FIG. 1  to  FIG. 8  show the automatic cell culture apparatus of the present invention, and  FIG. 9  to  FIG. 15  show the rotating culture vessel of the present invention. In Figures, the sign  1  represents the automatic cell culture apparatus, the sign  2  represents the rotating culture vessel, the sign  3  represents a rotating culture device, the sign  4  represents a cool supply box, the sign  5  represents a cool discharge box, the sign  6  represents a shaft direction shifting means, the sign  7  represents an XYZ-axis shifting mechanism for supply, and the sign  8  represents an XYZ-axis shifting mechanism for discharge. 
         [0056]    The automatic cell culture apparatus  1  of the present invention uses a rotating culture vessel  2  and is used for three-dimensional culture of cells in a microgravity environment that is nearly gravity-free. The automatic cell culture apparatus includes a closed housing  9  having an air conditioning function, the closed housing  9  includes a middle chamber that includes a rotating culture device  3  having an incubator box  10  in which one or more rotating culture vessels  2  are installed for culture, a lower chamber that includes a cool supply box  4  storing a supply syringe  11  for supplying a liquid culture medium, and an upper chamber that includes a cool discharge box  5  storing a discharge syringe  12  for collecting a liquid culture medium. Each of the incubator box  10 , the cool supply box  4 , and the cool discharge box  5  includes a front face having an automatically operated door (not shown in the drawings). The rotating culture device  3  has a shaft direction shifting means  6  shifting a horizontal rotating shaft  13  provided in the incubator box  10  back and forth in the shaft direction, and the rotating shaft  13  has an end that is removably installed with the rotating culture vessel  2 . A front space of the cool supply box  4  includes an XYZ-axis shifting mechanism  7  for supply, the XYZ-axis shifting mechanism  7  for supply includes a movable part for supply driven by the mechanism, and the movable part for supply has a fixing chuck  14  for holding the supply syringe  11  upward and a push up means  15  for pushing a piston  11 A up. A front space of the cool discharge box  5  includes an XYZ-axis shifting mechanism  8  for discharge, the XYZ-axis shifting mechanism  8  for discharge includes a movable part for discharge driven by the mechanism, and the movable part for discharge has a fixing chuck  16  for holding the discharge syringe  12  downward and a pull up means  17  for pulling a piston  12 A up. In the present embodiment, the shaft direction of the rotating shaft  13  is regarded as an X axis, the horizontal direction orthogonal to the X axis is regarded as a Y axis, and the vertical direction is regarded as a Z axis for description. 
         [0057]    In the present embodiment, a plurality of rotating shafts  13  that are shifted back and forth in the X axis direction by a common shaft direction shifting means  6  are provided while the rotating shafts are not overlapped in the vertical direction. Ends of the rotating shafts  13  are displaced back and forth so that the adjacent rotating culture vessels  2  will not interfere with each other when the rotating culture vessels  2  are installed to the corresponding rotating shafts  13 . In the present embodiment, two rotating shafts  13  are provided in parallel to achieve cell culture using two rotating culture vessels  2  at the same time. 
         [0058]    Next, the rotating culture vessel  2  will be described in detail based on  FIG. 9  to  FIG. 15 . The rotating culture vessel  2  includes a cell suspension supply port  19  for supplying a cell suspension composed of cells and a liquid culture medium on an outer circumferential cylindrical face of a flat cylindrical culture container  18  as well as includes an air bleeding port  20  and a cell discharge port  21  on a front face orthogonal to the rotating shaft  13  of the culture container  18 . The rotating culture vessel  2  further includes at least one pair of a supply port  22  and a discharge port  23  for liquid culture medium exchange on the outer circumferential cylindrical face of the culture container  18 . The pair of the supply port  22  and the discharge port  23  is positioned 180° opposite to each other, the discharge port  23  has a center line passing through the rotating center, and the supply port  22  has a center line eccentrically positioned with respect to the rotating center. 
         [0059]    In the present embodiment, three pairs of the supply ports  22  and the discharge ports  23  are provided on the outer circumferential cylindrical face of the culture container  18 , and these ports will be distinguished by A, B, and C. The supply ports  22 A,  22 B, and  22 C are provided at an equal angle interval and the discharge ports  23 A,  23 B, and  23 C are also provided at an equal angle interval. In the present embodiment, the cell suspension supply port  19  and the cell discharge port  21  are independently provided, but one port may be provided to serve as both ports. Here, the distance between the center line of the supply port  22  and the rotating center is 0.5r to 0.9r where the culture container  18  has a culture space having a radius of r. 
         [0060]    The rotating culture vessel  2  further includes, at the back center, a protruding attaching part  24  that is removably installed to the end of the rotating shaft  13  in the rotating culture device  3 . The attaching part  24  has a structure so that the vessel will be attached at the same rotational position with respect to the end of the rotating shaft  13  at any time. The rotating culture vessel  2  of the embodiment is expected to have a volume of 10 to 20 ml because a liquid culture medium is expensive, but the volume should be determined depending on the size of clumps of cells to be cultured. 
         [0061]    As shown in  FIG. 10 ,  FIG. 13 ,  FIG. 14 , and  FIG. 15 , the rotating culture vessel  2  has air intake ports  25  on the back side around the attaching part  24 , and a gas permeable membrane  26  is provided inside the air intake port  25 . Through the membrane, oxygen can be supplied into a liquid culture medium while carbon dioxide can be discharged. The rotating culture vessel  2  also has an observation window  27  on the front side so that the inside can be observed. 
         [0062]    As shown in  FIG. 15 , each of the supply port  22  and the discharge port  23  has a septum seal structure. The supply syringe  11  and the discharge syringe  12  used for liquid culture medium exchange have injection needles  11 B and  12 B, respectively, at the leading ends, and each injection needle can pass through the septum seal  28 . The supply port  22  is positioned downward, and the discharge port  23  is positioned upward. A new liquid culture medium is supplied from the lower side with the supply syringe  11  while a used liquid culture medium is sucked from the upper side with the discharge syringe  12  and air can be bled with the discharge syringe  12 . Each of the supply port  22  and the discharge port  23  has the septum seal structure, and thus the injection needles  11 B and  12 B installed to the leading ends of the supply syringe  11  and the discharge syringe  12  pass through the septum seals  28  to achieve airtight connection. Moreover, even when the injection needles  11 B and  12 B are removed, the through holes are closed due to elastic recovery of the septum seal to maintain the airtight condition. 
         [0063]    More specifically, the supply port  22  and the discharge port  23  have the same cross sectional structure as shown in  FIG. 15 . The supply port  22  includes a port  29  that is eccentrically positioned with respect to the rotating center of the outer circumferential cylindrical face of the culture container  18  and continues to the culture space. The supply port  22  further includes a silicon rubber septum seal  28  in an end part of the port  29 . The circumference of the septum seal  28  is pressed with a pressing cap  30  screwed with the port  29  to lead to a closed structure. Meanwhile, the discharge port  23  includes a port  29  that continues to the culture space toward the radial direction passing through the rotating center of the outer circumferential cylindrical face of the culture container  18 . The discharge port  23  further includes a silicon rubber septum seal  28  in an end part of the port  29 . The circumference of the septum seal  28  is pressed with a pressing cap  30  screwed with the port  29  to lead to a closed structure. Each port  29  constituting the supply port  22  and the discharge port  23  having the septum seal structures includes an inlet flow path  31  having a large diameter behind the septum seal  28  and an orifice flow path  32  having a small diameter between the inlet flow path  31  and the culture space in the culture container  18 , and the inlet flow path  31  accepts the injection needle  11 B or  12 B. 
         [0064]    The leading end of the injection needle  11 B of the supply syringe  11  passed thorough the septum seal  28  of the supply port  22  is accepted in the inlet flow path  31  in the port  29 , and thus even when the injection needle  11 B is inserted into the septum seal  28  slightly dislocated from the center, the leading end of the injection needle  11 B does not come in contact with the port  29  to be used without problems. Hence, request to the positioning precision of the XYZ-axis shifting mechanism  7  for supply is lowered, and the mechanism can be formed using a cheap actuator. The orifice flow path  32  is also provided. Thus, a liquid culture medium supplied in the inlet flow path  31  is introduced through the orifice flow path  32  having a small diameter into the culture space, and the disturbance generated in the culture space can be minimized. The leading end of the injection needle  12 B of the discharge syringe  12  passed through the septum seal  28  of the discharge port  23  is also accepted in the inlet flow path  31  in the port  29 , and a used liquid culture medium and air accumulated in the culture space can be discharged through the orifice flow path  32  by suction. Even when air is accumulated in the inlet flow path  31 , the air does not flow into the culture space through the orifice flow path  32  in rotation culture due to the surface tension of an liquid culture medium. 
         [0065]    As shown in  FIG. 14 , each of the cell suspension supply port  19  and the air bleeding port  20  has a septum seal structure similar to the above, and the cell discharge port  21  has a rubber plug structure having a large opening. That is, as with the discharge port  23 , the cell suspension supply port  19  includes a port  29  that continues to the culture space toward the radial direction passing through the rotating center. The cell suspension supply port  19  also includes a silicon rubber septum seal  28  in an end part of the port  29 . The circumference of the septum seal  28  is pressed with a pressing cap  30  screwed with the port  29  to lead to a closed structure. The air bleeding port  20  and the cell discharge port  21  are positioned on the periphery of the front face of the culture container  18  opposite to each other with respect to the rotating center. The air bleeding port  20  includes a port  33  protruded from the front face of the culture container  18  and a silicon rubber septum seal  34  in an end part of the port  33 . The circumference of the septum seal  34  is pressed with a pressing cap  35  screwed with the port  33  to lead to a closed structure. Each of the port  29  of the cell suspension supply port  19  and the port  33  of the air bleeding port  20  includes an inlet flow path  31  and an orifice flow path  32  in a similar manner to the above. An inside where the orifice flow path  32  of the air bleeding port  20  is opened toward the culture space has a concave part  36  in which air in the culture space is accumulated when the rotating culture vessel  2  is horizontalized. The cell discharge port  21  includes a port  37  having a large inner diameter, and the port is tightly fitted with a rubber plug  38 . The rubber plug  38  has a leading end face that is flush with the inner wall of the culture space so as not to disturb a liquid culture medium in rotation culture. 
         [0066]    In order to supply cells and a liquid culture medium into the rotating culture vessel  2 , the rotating culture vessel  2  is left with the observation window  27  facing upward and the rotating shaft in the vertical direction, an injection needle of a cell suspension supply syringe (not shown in the drawings) containing the cells and the liquid culture medium is inserted into the septum seal  28  of the cell suspension supply port  19 , an injection needle of an empty air bleeding syringe (not shown in the drawings) is inserted into the septum seal  34  of the air bleeding port  20 , and the cell suspension is supplied from the cell suspension supply syringe into the culture space while air is bled with the air bleeding syringe. At the time, the air in the culture space is finally accumulated in the concave part  36 , and the air accumulated in the concave part  36  is discharged from the culture space. 
         [0067]    Next, the automatic cell culture apparatus  1  for culturing cells while automatically exchanging a liquid culture medium using the rotating culture vessel  2  will be described in detail based on  FIG. 1  to  FIG. 8  and  FIG. 16  to  FIG. 18 . As shown in  FIG. 1 , the closed housing  9  has opening and closing doors on the front face and one side face. The front face has three inspection doors  39  corresponding to the rotating culture device  3 , the cool supply box  4 , and the cool discharge box  5 . The closed housing  9  has a transparent front panel so that the inside can be observed. The closed housing  9  includes the side face having one operation door  40  for the preparation of a series of culture or for the treatment after culture. That is, the operation door  40  is opened, the rotating culture vessel  2  is attached to or removed from the rotating shaft  13 , the supply syringes  11  are attached to or removed from the inside of the cool supply box  4 , and the discharge syringes  12  are attached to or removed from the inside of the cool discharge box  5 . The operation door  40  is transparent so that the inside can be observed. The closed housing  9  includes a ceiling having an air conditioner  41  with filter function for keeping the inside at suitable temperature and for air cleaning. 
         [0068]    As shown in  FIG. 1  to  FIG. 4 , the rotating culture device  3  includes the incubator box  10  in which the temperature can be controlled and that contains a horizontal rotating shaft  13  as well as includes a rotation control mechanism  42  behind the incubator box, for driving the rotating shaft  13  at a predetermined rotation speed, and a shaft direction shifting means  6  for shifting the rotating shaft  13  together with the rotation control mechanism  42  back and forth in the shaft direction. The incubator box  10  has an automatically operated door on the front face and a manually operated door  43  on the side face having the operation door  40 . In the present embodiment, two rotating shafts  13  are arranged in parallel in a horizontal position, and each rotating shaft is supported with a cylindrical shaft bearing  44  rotatably and slidably in the shaft direction. The shaft direction shifting means  6  has a structure to shift a movable part  46  in front and back directions along a linear guide  45  provided behind the incubator box  10 . Ends of the rotating shafts  13  continuing backward from the back face of the incubator box  10  are interlocked by a rotation control mechanism  42  that is attached to the movable part  46  of the shaft direction shifting means  6  through a timing belt. There are two independent rotation control mechanisms  42  so as to independently control each rotation of the rotating shafts  13 . The rotation control mechanism  42  is driven by a stepping motor or a servomotor so as to accurately control the rotation speed and the rotational position and controls the rotational position by, for example, reading a marker fixed on the rotating shaft  13  with a sensor. The leading end positions of the rotating shafts  13  are displaced back and forth as described above, and the rotating culture vessel  2  filled with a cell suspension is manually attached to or removed from each leading end. 
         [0069]    As shown in  FIG. 1  to  FIG. 3  and  FIG. 7 , the cool supply box  4  includes an automatically operated door on the front face of the box in which temperature can be controlled, a manually operated door  47  on the side face having the operation door  40 , and a rotating revolver type stacker  48  in the cool supply box. The stacker  48  includes a vertical rotating shaft  50  that is controlled with a stepping motor  49  and a plurality of holders  51  around the stacker  48 . The holder  51  elastically removably holds the supply syringe  11  in a transverse direction with the injection needle  11 B facing upward in the vertical direction. The holder  51  of the stacker  48  include an U-groove that accepts and locks the upper and lower parts of the supply syringe  11  and a grip that elastically grips the supply syringe  11  from both sides for holding. The supply syringe  11  is laterally pushed to be automatically held with the holder, and the supply syringe  11  is grasped and laterally pulled from the holder to be readily removed. 
         [0070]    As shown in  FIG. 1  to  FIG. 3  and  FIG. 8 , the cool discharge box  5  has approximately the same structure as that of the cool supply box  4 , and includes an automatically operated door on the front face of the box in which temperature can be controlled, a manually operated door  52  on the side face having the operation door  40 , and a rotating revolver type stacker  53  in the cool discharge box  5 . The stacker  53  includes a vertical rotating shaft  55  that is controlled with a stepping motor  54  and a plurality of holders  56  around the stacker  53 . The holder elastically removably holds the discharge syringe  12  in a transverse direction with the injection needle  12 B facing downward in the vertical direction. The stacker  53  has approximately the same structure as that of the stacker  48 . 
         [0071]    Next, the XYZ-axis shifting mechanism  7  for supply for transferring the supply syringe  11  will be described based on  FIG. 2 ,  FIG. 3 , and  FIG. 5 . The XYZ-axis shifting mechanism  7  for supply is provided in a front space of the cool supply box  4  in the closed housing  9 . The XYZ-axis shifting mechanism  7  for supply includes a Y-axis shifting mechanism  57  fixed to the bottom in the closed housing  9 , a Z-axis shifting mechanism  58  fixed to a movable part of the Y-axis shifting mechanism  57 , and an X-axis shifting mechanism  59  fixed to a movable part of the Z-axis shifting mechanism  58 . The X-axis shifting mechanism  59  includes a movable part having a fixing chuck  14  for holding the supply syringe  11  upward and having a push up means  15  for pushing a piston  11 A up. Each of the Y-axis shifting mechanism  57 , the Z-axis shifting mechanism  58 , and the X-axis shifting mechanism  59  is composed of a linear guide and a stepping motor driven with a ball screw, but the structure is not specifically limited. The fixing chuck  14  is composed of a U-groove plate  60  that locks a flange part of a cylinder of the supply syringe  11  and an air-driven hand  61  that grips the side face of the cylinder. The push up means  15  includes a protruded push up plate  63  that is provided on a movable part of a Z-axis shifting mechanism  62  and that is in contact with the lower end of the piston  11 A. The movable part of the Z-axis shifting mechanism  62  along with the fixing chuck  14  are fixed to the movable part of the X-axis shifting mechanism  59 . 
         [0072]    Finally, the XYZ-axis shifting mechanism  8  for discharge for transferring the discharge syringe  12  will be described based on  FIG. 2 ,  FIG. 3 , and  FIG. 6 . The XYZ-axis shifting mechanism  8  for discharge is provided in a front space of the cool discharge box  5  in the closed housing  9 . The XYZ-axis shifting mechanism  8  for discharge includes a Y-axis shifting mechanism  64  fixed to an anterior top area in the closed housing  9 , a Z-axis shifting mechanism  65  fixed to a movable part of the Y-axis shifting mechanism  64 , and an X-axis shifting mechanism  66  fixed to a movable part of the Z-axis shifting mechanism  65 . The X-axis shifting mechanism  66  includes a movable part having a fixing chuck  16  for holding the discharge syringe  12  downward and a pull up means  17  for pulling a piston  12 A up. The fixing chuck  16  is composed of a U-groove plate  67  that locks a flange part of a cylinder of the discharge syringe  12  and an air-driven hand  68  that grips the side face of the cylinder. The pull up means  17  include a protruded pull up plate  70  that is provided on a movable part of a Z-axis shifting mechanism  69  and that locks a flange part at the upper end of the piston  12 A for pulling up. The movable part of the Z-axis shifting mechanism  69  along with the fixing chuck  16  are fixed to the movable part of the X-axis shifting mechanism  66 . 
         [0073]    The procedure for culturing cells using the automatic cell culture apparatus  1  of the present invention will be described below. First, the operation door  40  of the closed housing  9  is opened, the door  47  of the cool supply box  4  is opened, a predetermined number of supply syringes  11  are installed to the stacker  48 , and the door  47  is closed. The door  52  of the cool discharge box  5  is opened, an equal number of the discharge syringes  12  to that of the supply syringes  11  are installed to the stacker  53 , and the door  52  is closed. Meanwhile, the door  43  of the incubator box  10  of the rotating culture device  3  is opened, the attaching part  24  of the rotating culture vessel  2  filled with a cell suspension is installed to the end of the rotating shaft  13 , and the door  43  is closed. At the time, the rotating culture vessel  2  is fixed to a precise rotational position with respect to the rotating shaft  13  at any time. Next, the operation door  40  is closed, and the temperature in the closed housing  9  together with the incubator box  10  is kept at a predetermined temperature. This prevents that the temperature in the incubator box  10  is steeply changed to change a culture condition when the automatically operated door on the front face of the incubator box  10  is opened for liquid culture medium exchange. Each temperature in the cool supply box  4  and the cool discharge box  5  is controlled to be lower than the temperature in the incubator box  10  in order to store a new liquid culture medium before use and a used liquid culture medium without varying the condition. 
         [0074]    Then, the rotation control mechanism  42  is driven to rotate the rotating culture vessel  2  at a predetermined rotation speed for cell culture. After culturing for a predetermined period of time, the rotation control mechanism  42  is controlled to stop the rotating culture vessel  2  with the first supply port  22 A facing downward in the vertical direction and the first discharge port  23 A facing upward in the vertical direction. Next, the automatically operated door on the front face of the incubator box  10  is opened, the shaft direction shifting means  6  is driven to shift the rotating culture vessel  2  forward, and the rotating culture vessel  2  is located in a front space of the incubator box  10 . Simultaneously or before or after that, each automatically operated door on the front faces of the cool supply box  4  and the cool discharge box  5  is opened, each of the XYZ-axis shifting mechanism  7  for supply and the XYZ-axis shifting mechanism  8  for discharge is independently driven, the fixing chuck  14  is transferred into the cool supply box  4 , the supply syringe  11  stored in the stacker  48  at a predetermined rotational position is held with the fixing chuck  14 , then the supply syringe  11  is transferred into a front space of the cool supply box  4 , as well as the fixing chuck  16  is transferred into the cool discharge box  5 , the discharge syringe  12  stored in the stacker  53  at a predetermined rotational position is held with the fixing chuck  16 , and then the discharge syringe  12  is transferred into a front space of the cool discharge box  5 . 
         [0075]    Next, the supply syringe  11  is located directly below the first supply port  22 A of the rotating culture vessel  2 , while the discharge syringe  12  is located directly above the discharge port  23 A. Then, as shown in  FIG. 16 , the Z-axis shifting mechanism  58  of the XYZ-axis shifting mechanism  7  for supply and the Z-axis shifting mechanism  65  of the XYZ-axis shifting mechanism  8  for discharge are synchronously driven, the injection needle  11 B of the supply syringe  11  is passed through the septum seal  28  of the supply port  22 A for connection, and simultaneously, the injection needle  12 B of the discharge syringe  12  is passed through the septum seal  28  of the discharge port  23 A for connection. While maintaining this condition, the push up plate  63  of the push up means  15  is elevated to push the piston  11 A of the supply syringe  11  up, a new liquid culture medium is supplied into the culture space of the rotating culture vessel  2 , simultaneously, the pull up plate  70  of the pull up means  17  is elevated to pull the piston  12 A of the discharge syringe  12  up, a used liquid culture medium is sucked out from the culture space of rotating culture vessel  2 , and at the time, air accumulated in the culture space during culturing is also simultaneously sucked out. After the liquid culture medium in the rotating culture vessel  2  is exchanged in this manner, the Z-axis shifting mechanism  58  of the XYZ-axis shifting mechanism  7  for supply and the Z-axis shifting mechanism  65  of the XYZ-axis shifting mechanism  8  for discharge are synchronously driven, the injection needle  11 B of the supply syringe  11  is removed from the supply port  22 A, and simultaneously, the injection needle  12 B of the discharge syringe  12  is removed from the discharge port  23 A. Next, the XYZ-axis shifting mechanism  7  for supply and the XYZ-axis shifting mechanism  8  for discharge are driven, the used supply syringe  11  and the used discharge syringe  12  are installed to the original positions in the stacker  48  and the stacker  53 , respectively, the fixing chuck  14  and the fixing chuck  16  are unlocked and returned from the cool supply box  4  and the cool discharge box  5  to stay at the initial positions. Meanwhile, the shaft direction shifting means  6  is driven to store the rotating culture vessel  2  in the incubator box  10 , each automatically operated door is closed, and then the rotation control mechanism  42  is driven to rotate the rotating culture vessel  2  at a predetermined rotation speed for cell culture. 
         [0076]    The liquid culture medium exchange operation is performed using a pair of the supply port  22 B and the discharge port  23 B of the rotating culture vessel  2  as shown in  FIG. 17 , and then the operation is repeated using a pair of the supply port  22 C and the discharge port  23 C as shown in  FIG. 18 . In this case, each of the stacker  48  and the stacker  53  is rotated by a predetermined angle so that a new supply syringe  11  and a new discharge syringe  12  will be located at the front in sequence. 
         [0077]    The rotating culture device  3  employed in the present invention can keep cells in suspension without settling in the rotating culture vessel  2 . Therefore, the rotating culture device  3  has advantages that three-dimensional aggregates can be formed, necrosis due to stirring stress can be avoided, differentiation inducers effectively work, and removal of waste products and supply of nutrients can be performed. In the present invention, a liquid culture medium can be sequentially supplied using a plurality of the supply syringes  11 . Hence, a liquid culture medium having an optimum composition may be used depending on a culture stage of cells. 
         [0078]    It is important to observe a culture condition in culture. The observation of the culture condition is carried out on, for example, (1) pH change and color change of a culture medium due to the consumption of culture medium additives, accumulation of waste products, and the like in culture, (2) presence or absence of turbidity of a culture medium due to contamination, and (3) whether a three-dimensional tissue is formed from floating cells. In the present invention, the rotating culture vessel  2  has the observation window  27  on the front face. Thus, the inner condition can be observed through an imaging camera or various analytical equipments placed toward the observation window  27 , and an actual condition can be analyzed through image processing. Based on the condition, the rotation control mechanism  42  can be feedback-controlled, and the timing of liquid culture medium exchange can be automatically determined. 
       EXAMPLES 
       [0079]    Next, rotation cultures were performed using the automatic cell culture apparatus of the present invention and by hand as a control (using RCCS-4D manufactured by Synthecon, the rotation speed was visually controlled). Cartilage tissue formation experiment was carried out using bone marrow cells of Japanese white rabbit to compare the both rotation cultures. The experimental procedure will be described below. 
         [0080]    (Experimental Procedure) 
         [0081]    (1) Bone marrow cells were collected from the long bones of two Japanese white rabbits, 10 days old, and suspended in 20 ml standard medium. 
         [0082]    *Standard Medium: DMEM (Dulbeccco&#39;s modified Eagle&#39;s medium (DMEM, Sigma, St. Louis Mo.)+10% FBS (fetal bovine serum)+antibiotic-antimycotic (Invitrogen, Carlsbad, Calif.) 
         [0083]    (2) Next, the cells were seeded into a 75T flask (BD) together with 15 ml standard medium and cultured in 5% CO2 at 37° C. for 3 weeks. 
         [0084]    (3) Next, the cells were removed with trypsin, suspended in a bioreactor medium, and transferred into a 50 cc vessel. 
         [0085]    *Bioreactor Medium: DMEM+50 μg/ml ascorbic acid (WAKO)+40 μg/m1L-proline+ITS culture supplement (BD Biosciences), 10-7 dexamethasone (Sigma), 10 ng/ml TGF-β3 (Sigma), and abtibiotic-antimycotic (BD) 
         [0086]    *The 50 cc vessel used in Example is that shown in  FIG. 9  to  FIG. 15 . 
         [0087]    *The number of cells used for the culture using the automatic cell culture apparatus (Example) was the same as that for the manual culture (Comparative Example). 
         [0088]    (4) The cells were cultured for 2 weeks. The tissue was taken out, macroscopically observed, and sliced into sections. The sections were evaluated by histochemical techniques. 
         [0089]    During the liquid culture medium exchange by the automatic cell culture apparatus of the present invention, cellular tissues were not hit by the vessel wall. Furthermore, the liquid culture medium did not leak between each injection needle of the supply syringe and the discharge syringe and the septum seal. It could be observed that the used liquid culture medium in the vessel was exchanged with the new liquid culture medium in sequence from the bottom. There was no accumulated gas in the upper part of the vessel after the liquid culture medium exchange. 
         [0090]    (Culture Result) 
         [0091]      FIG. 19  shows the appearances of the cartilage tissues formed by the cultures. In  FIG. 19 , the left image shows the result of the automatic culture (Example) and the right image shows the result of the manual culture (Comparative Example). Macroscopic observations of the cultured cartilage tissues revealed that the tissue by the manual culture was larger than that by the automatic culture. 
         [0092]      FIG. 20  is a graph showing the result comparing the production amounts of GAG in the cartilage matrix. In this culture, the production amount by the automatic culture was higher. In the experiments repeated several times, the production amount of GAG in the cartilage matrix by the automatic culture was the same as or higher than that by the manual culture. 
         [0093]    Finally,  FIGS. 21  to  FIGS. 23  show the results of the histological evaluations on the cartilage tissues formed by the cultures.  FIGS. 21  are micrographs showing the results of the cartilage tissues stained with Alcian blue. In both tissues, the cartilage matrix was stained light blue, and the rich production of the cartilage matrix was confirmed. Here, the area where the cartilage matrix was stained light blue is indicated by dark color in  FIGS. 21 . 
         [0094]      FIGS. 22  are micrographs showing the results of the cartilage tissues stained with toluidine blue. In both tissues, the cartilage matrix was stained blue purple, and the rich production of the cartilage matrix was confirmed. In this case, the area where the cartilage matrix was stained blue purple is also indicated by dark color in  FIGS. 22 . 
         [0095]      FIGS. 23  are micrographs showing the results of the cartilage tissues stained with HE (hematoxylin-eosin). The cartilage tissue was stained blue purple with hematoxylin, and the mature chondrocytes stained blue purple were observed in both tissues by the automatic culture and the manual culture. In this case, the area stained blue purple is also indicated by dark color in  FIGS. 23 . However, cytoplasm, connective tissue in soft tissue, red blood cells, fibrin, endocrine granules, and the like are stained light red or indigo blue with eosin. Thus, each stained area is indicated by similar dark color in a monochrome image, and the cartilage tissue cannot be distinguished in the monochrome image. 
         [0096]    As described above, the culture using the automatic cell culture apparatus of the present invention was compared with that by hand while controlling the rotation. The result of the culture of rabbit bone marrow cells with the rotating culture device using the RWV vessel revealed that the automatic culture was equal or superior in quality to the manual culture. 
       INDUSTRIAL APPLICABILITY 
       [0097]    According to the automatic cell culture apparatus of the present invention, even medical institutions not having cell processing center (CPC) in compliance with good manufacturing practice (GMP) can expect clinical application of regenerative medicine, and consequently, the regenerative medicine can be greatly generalized. Typically, it can be used in order to form transplantable cartilage tissues from human bone marrow cells. In addition to the cartilage regeneration, the study of the regenerative medicine has been extended to corneal regeneration for retinal detachment, cataract, and the like, bone regeneration for bone defect and osteoporosis, pancreas (Langerhans island) regeneration for diabetes mellitus and the like, cardiac muscle regeneration for dilated cardiomyopathy and the like, nerve regeneration for Parkinson&#39;s disease and Alzheimer&#39;s disease, and the like. Hence, the automatic cell culture apparatus of the present invention is supposed to have an advantage in the regenerative medicine in addition to the cartilage regeneration. The automatic cell culture apparatus of the present invention will be generally applicable to the regenerative medicine in addition to the cartilage regenerative medicine in future, and will certainly be the essential and important basic technique for the generalization of the regenerative medicine. 
       Reference Signs List 
       [0098]      1  Automatic cell culture apparatus 
         [0099]      2  Rotating culture vessel 
         [0100]      3  Rotating culture device 
         [0101]      4  Cool supply box 
         [0102]      5  Cool discharge box 
         [0103]      6  Shaft direction shifting means 
         [0104]      7  XYZ-axis shifting mechanism for supply 
         [0105]      8  XYZ-axis shifting mechanism for discharge 
         [0106]      9  Closed housing 
         [0107]      10  Incubator box 
         [0108]      11  Supply syringe 
         [0109]      11 A Piston 
         [0110]      11 B Injection needle 
         [0111]      12  Discharge syringe 
         [0112]      12 A Piston 
         [0113]      12 B Injection needle 
         [0114]      13  Rotating shaft 
         [0115]      14  Fixing chuck 
         [0116]      15  Push up means 
         [0117]      16  Fixing chuck 
         [0118]      17  Pull up means 
         [0119]      18  Culture container 
         [0120]      19  Cell suspension supply port °Air bleeding port &lt;Cell discharge port 
         [0121]      22 ,  22 A,  22 B,  22 C Supply port 
         [0122]      23 ,  23 A,  23 B,  23 C Discharge port 
         [0123]      24  Attaching part 
         [0124]      25  Intake port 
         [0125]      26  Gas permeable membrane 
         [0126]      27  Observation window 
         [0127]      28  Septum seal 
         [0128]      29  Port 
         [0129]      30  Cap 
         [0130]      31  Inlet flow path 
         [0131]      32  Orifice flow path 
         [0132]      33  Port 
         [0133]      34  Septum seal 
         [0134]      35  Cap 
         [0135]      36  Concave part 
         [0136]      37  Port 
         [0137]      38  Rubber plug 
         [0138]      39  Inspection door 
         [0139]      40  Operation door ″Air conditioner 
         [0140]      42  Rotation control mechanism 
         [0141]      43  Door 
         [0142]      44  Shaft bearing 
         [0143]      45  Linear guide 
         [0144]      46  Movable part 
         [0145]      47  Door 
         [0146]      48  Stacker 
         [0147]      49  Stepping motor 
         [0148]      50  Rotating shaft 
         [0149]      51  Holder 
         [0150]      52  Door 
         [0151]      53  Stacker 
         [0152]      54  Stepping motor 
         [0153]      55  Rotating shaft 
         [0154]      56  Holder 
         [0155]      57  Y-axis shifting mechanism 
         [0156]      58  Z-axis shifting mechanism 
         [0157]      59  X-axis shifting mechanism 
         [0158]      60  U-groove plate 
         [0159]      61  Hand 
         [0160]      62  Z-axis shifting mechanism 
         [0161]      63  Push up plate 
         [0162]      64  Y-axis shifting mechanism 
         [0163]      65  Z-axis shifting mechanism 
         [0164]      66  X-axis shifting mechanism 
         [0165]      67  U-groove plate 
         [0166]      68  Hand 
         [0167]      69  Z-axis shifting mechanism 
         [0168]      70  Pull up plate