JP5958861B2 - Incubator - Google Patents

Description

  The present invention relates to a culture device and a culture device system including the culture device.

  Since pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can be differentiated into cells of any tissue or organ in the body, research and development have been actively conducted in recent years. Yes. Since these pluripotent stem cells can be a new cell source for cell therapy, expectations for clinical application are increasing. However, because pluripotent stem cells have the ability to differentiate into cells of any tissue, it is difficult to amplify and culture in large quantities while maintaining an undifferentiated state, and a culture method different from conventional culture methods is necessary. As a method for carrying out amplification culture while maintaining the undifferentiated state of pluripotent stem cells, a method is known in which a cell aggregate called an embryoid body (EB) is formed and cultured. This is a culture method in which a cell mass is formed so that a vertebrate mimics an embryo formed by repeated cleavage after fertilization. By forming an embryoid body, it is possible to mimic embryonic development and induce differentiation into any cell by adding various growth factors and the like.

  In order to induce differentiation of ES cells and iPS cells, it is necessary to go through the process of embryoid body formation. There are various methods for the formation of embryoid bodies. For example, a method using a low-adhesion culture dish, a method called a hanging drop method, a method of attaching a culture solution containing cells to the lid of a culture dish, a method of suspension stirring culture, and the like can be mentioned. The three-dimensional culture by floating agitation can control the process and is suitable for scale-up. On the other hand, since human-derived ES / iPS cells are particularly sensitive to shear stress, a means of stirring with low shear stress is required in suspension stirring culture. Therefore, for example, measures such as reducing the blade area of the stirring blade and suppressing the tip speed of the blade during stirring are taken. In embryoid body formation, ES cells and iPS cells proliferate in the embryoid body while maintaining undifferentiation, and the specific gravity of the embryoid body increases. The target cells cannot be obtained in high yield due to culture. The main cause is that the embryoid bodies aggregate at a location where the liquid flow in the culture tank is non-uniform, and the specific gravity increases, so that floating agitation itself does not hold.

  In addition, embryoid bodies with a particle size that is too large, cells inside the embryoid body fall into an ischemic (hypotrophic, hypoxic) state and promote cell death. It also hinders uniform access (diffusion) to the inside. When cell aggregates with non-uniform particle sizes are formed, nutrients, oxygen, differentiation-inducing factors, etc. added during cell differentiation cannot penetrate into the cell aggregates, causing cell growth. And will inhibit differentiation induction.

  As a culture method from the viewpoint of liquid flow, conventionally, a method of creating a flow perpendicular to the stirring shaft using a stirring blade composed of a plurality of blades attached at a fixed inclination angle to the stirring shaft (shaft (Flow culture) and several large paddles standing vertically with respect to the bottom of the culture tank and rotating a valve-shaped stirring shaft with a thick tip to create a horizontal flow (when viewed from above, donut-shaped) The method (laminar flow culture) was common. In axial flow culture, a large number of stirring blades are required to uniformly stir the entire culture tank at a low rotation, which may increase the shear stress due to the rotation. In addition, there is a problem that it is easy to stagnate just below the axis due to the structure, and cell aggregates (embryoid bodies, etc.) having increased specific gravity with the growth of the cells precipitate and it is difficult to obtain cell aggregates of uniform particle size. In laminar flow culture, if the rotational speed is set so as not to cause turbulent flow, it is easy to achieve stirring with extremely low shear stress, but there is a problem that the central part of the donut-shaped liquid flow is likely to stagnate.

  Therefore, the applicant of the present application enables laminar flow culture with low shear stress and no stagnation of the liquid flow in the culture tank, particularly without stagnation in the vicinity of the center of the bottom in cell culture. Has developed a cell culture device that can easily and reproducibly obtain a population of cell aggregates (such as embryoid bodies) (Japanese Patent Application No. 2012-131597).

According to the cell culture apparatus as described above, the liquid flow in the culture tank can be made uniform. On the other hand, in the culture apparatus, a measuring instrument (measuring means) for measuring the characteristics of dissolved oxygen (DO), pH, temperature, dissolved carbon dioxide gas, etc. in the culture solution in the culture tank in order to maintain an optimal culture state. ) Is provided. Conventionally, in order to measure the above-described characteristics in a culture solution in a culture tank, for example, in the case of dissolved oxygen, a galvanic cell type or polarographic type is used, and in the case of pH, a rod-shaped sensor such as a glass composite electrode is directly used. Inserting into a culture tank is performed. As a conventional measuring instrument used for such a technique, a rod-type sensor such as an electrode type, which is sold by, for example, Hamilton of the United States, is known.

  However, in order to cultivate human iPS cells and the like in a small volume of culture solution having a culture volume of several tens to 100 mL, in such a conventional method, an excluded volume (ie, Since the volume of the portion where the sensor is immersed in the culture solution is too large, there is a possibility that uniform stirring in the culture tank may be hindered. Therefore, in order to achieve uniform stirring in the culture tank, it is necessary not only to devise the shape of the culture tank and the stirring blade, but also to reduce the size of various sensors to the extent that they do not interfere with uniform stirring.

  In order to reduce the size of the sensor, it may be possible to review the design of the conventional rod-shaped sensor, introduce an optical measurement method, etc. This method is desirable. As such a sensor, for example, a patch type (spot) fluorescent probe commercially available from Presense, Germany (a part of measuring means, a luminescent substance having a property that the fluorescent characteristic changes depending on dissolved oxygen and pH (fluorescent substance) ) Is applied to the fluorescent probe (patch type) (for example, a thin piece having an appropriate shape such as a circle), and the fluorescent probe is irradiated with light and received from the fluorescent probe. There is known a system (sensor) in which a measuring instrument (a part of measuring means) provided with a light emitting / receiving unit or the like is a separate component. This patch-type fluorescent probe is constituted by a technique such as applying a luminescent substance (fluorescent substance) to the surface of a transparent glass plate, a resin plate, or a transparent resin film. The sheet surface is attached to the inner wall of the culture tank so that the sheet surface is located below the liquid level in the culture liquid in the culture tank. When light is irradiated from the outside of the pasting surface through a transparent culture vessel wall and an optical fiber, the luminescent material is excited to emit fluorescence, and this fluorescence is sent to the measuring instrument arranged outside the culture vessel through the optical fiber. By measuring the intensity (phase angle) of fluorescence with a measuring instrument, it is possible to measure characteristics such as dissolved oxygen concentration, pH, and dissolved carbon dioxide gas. According to this system, a nozzle for inserting and holding a conventional rod-shaped sensor as described above into a culture tank as long as the culture tank is transparent and the fluorescent probe can be attached to the inside. Or a connector is unnecessary.

  In addition, in the culture of human iPS cells, for which future clinical application is expected, it is essential to use a single-use culture tank (one-time use). The fluorescent probe is attached to the inner wall surface of the culture tank in advance.

  However, when the patch-type fluorescent probe as described above is used, the user attaches a fluorescent probe to the culture vessel when using the culture vessel, or uses a culture vessel on which the fluorescent probe is attached in advance. In any case, the positional relationship between the attached fluorescent probe and the tip of the optical fiber affects the measurement accuracy, so an optical fiber is used to reliably receive the fluorescence through the culture vessel wall and the optical fiber outside the surface to which the fluorescent probe is attached. Must be placed at an appropriate position and at an appropriate angle with respect to the fluorescent probe. In other words, the distance between the fluorescent probe attachment surface and the tip of the optical fiber is within a few millimeters, and the tip of the optical fiber is always in close contact with the outer wall of the culture vessel in order to prevent irregular reflection of light on the outer wall of the culture vessel. A jig to do this must be prepared.

  On the other hand, such a simple fluorescent probe can be used once and can be disposed of in a sterilized state in combination with a disposable culture tank, thereby greatly reducing the time required for user preparation for culture. be able to. However, because the fluorescent probe has a time limit for maintaining the initial fluorescence characteristics after it is manufactured, the so-called expiration date is set, so the expiration date of the culture vessel in which the fluorescent probe is attached in advance is limited by the expiration date of the fluorescent probe. There is a problem of being done. In addition, it is impossible to exchange only the fluorescent probe during use of the culture tank.

  The present invention has been made to solve the above-mentioned problems, minimizes the volume excluded in the culture vessel by the sensor, and makes it very easy to exchange the fluorescent probe. A culture apparatus capable of easily performing relative positioning between the probe and the tip of an optical fiber that irradiates light to the fluorescence probe and receives fluorescence from the fluorescence probe, and the culture apparatus An object is to provide a culture apparatus system.

  In order to solve the above-described problems, the present invention provides a culture apparatus in which a measuring means for measuring a predetermined characteristic of a culture solution is detachably connected to a culture tank via an optical fiber. In this culture apparatus, the culture tank has a substantially cylindrical shape as a whole, the culture tank includes a generally cylindrical port extending radially outward from the side wall of the culture tank, The longitudinal axis of the port is arranged so as to be perpendicular to the central axis of the substantially cylindrical culture tank, and the culture device has an optical fiber receiving portion for removably receiving the optical fiber of the measuring means. A sensor connector that can be fitted to a port, and has a cylindrical shape as a whole, having two sections of a cylindrical tip side portion and a base end side portion, and the tip side portion is an outer outer cylinder portion And a sensor connector having an inner cylinder portion disposed inside the outer cylinder portion. The port of the culture tank has an inner end on the side wall side of the culture tank, an outer end opposite to the inner end, and an inner peripheral surface defining a passage for receiving the sensor connector. A thread projecting outward in the radial direction is formed in a portion adjacent to the end, and the inner peripheral surface of the port is formed so as to taper from the outer end toward the inner end. The outer peripheral surface of the inner cylindrical portion of the sensor connector is formed to taper toward the tip of the inner cylindrical portion. On the inner peripheral surface of the outer cylindrical portion of the sensor connector, a thread that can be screw-engaged with the thread of the port is formed, and the tip of the inner cylindrical portion of the sensor connector is closed by a tip wall portion. The tip wall is perpendicular to the center axis of the sensor connector, and on the outer surface of the tip wall is a fluorescent material that emits fluorescence corresponding to the predetermined characteristics of the culture solution by light irradiated through the optical fiber. A fluorescent probe is fixedly attached, and a stopper for holding the optical fiber in the sensor connector is provided in a portion of the sensor connector farther from the culture tank than the inner cylinder part and the outer cylinder part.

  The length of the inner cylinder part of the sensor connector is such that the tip wall part of the inner cylinder part is flush with the inner surface of the side wall of the culture tank when the sensor connector is attached to the port. Is preferred.

  It is preferable that the fluorescent probe fixed to the outer surface of the tip wall portion of the sensor connector has a patch shape (patch type).

  The port may be formed as a female luer connector and the sensor connector may be formed as a male luer connector.

  Further, the culture apparatus as described above, and a measuring means having a light emitting and receiving unit for irradiating the fluorescent probe of the culture apparatus with light through an optical fiber and receiving fluorescence emitted from the fluorescent probe, It is also possible to configure a culture apparatus system.

  According to the present invention having the above-described configuration, when the sensor connector having the fluorescent probe attached to the tip wall portion is attached to the port of the culture tank, the position of the tip wall portion of the sensor connector relative to the inner wall surface of the culture tank varies depending on the individual. Since the structure is always substantially uniform, it is possible to substantially eliminate the excluded volume of the fluorescent probe in the culture tank.

  In addition, the tip of the optical fiber when inserted until the optical fiber hits the inner tube of the sensor connector from the optical fiber inlet disposed on the side opposite to the tip wall of the sensor connector is the tip of the sensor connector. It is possible to fix the position of the distal end portion of the optical fiber with respect to the fluorescent probe attached to the distal end portion of the sensor connector, with the stopper provided on the sensor connector accurately disposed on the inner wall surface of the wall portion.

  Furthermore, since the fluorescent probe can be detached from the culture tank together with the sensor connector, it is possible to provide the fluorescent probe and the culture tank individually, and inventory management of components with greatly different expiration dates can be performed. become able to.

It is an external appearance perspective view which shows the whole culture tank (illustration of a cover part is abbreviate | omitted) to which the sensor connector of this invention is applied. 2A is a vertical cross-sectional view of the culture tank (the lid portion is not shown) shown in FIG. 1, FIG. 2B is a front view, and FIG. 2C is a top view. It is a longitudinal cross-sectional view which shows the culture apparatus of the state which provided the stirring means and the sensor connector in the culture tank shown in FIG. It is the front view (A) and sectional drawing (B) of the sensor connector of this invention. It is a cross-sectional perspective view which shows the state which fixed the fluorescent probe to the sensor connector and attached the optical fiber.

  A mode for carrying out the present invention will be described below with reference to the drawings.

  FIG. 1 is a diagram showing a culture tank 10 (a lid portion is not shown) of a culture apparatus 1 to which a sensor connector according to this embodiment is applied. 2A is a longitudinal sectional view of the culture tank shown in FIG. 1, FIG. 2B is a front view, and FIG. 2C is a top view. FIG. 3 is a longitudinal sectional view showing the culture apparatus in a state where the agitation means and the sensor connector are attached to the culture tank shown in FIG.

  The culture apparatus 1 of the present invention includes a fluorescent probe (a part of the measuring means) for measuring predetermined characteristics of a culture solution, for example, dissolved oxygen, pH, temperature, dissolved carbon dioxide gas, etc., via an optical fiber, and this fluorescent probe. A light viver (part of the measuring means) connected to the light emitting / receiving unit that receives the fluorescence emitted from the fluorescent probe and receives the fluorescence emitted from the fluorescent probe is detachably (detachably) connected to the culture tank 10. is there.

  The culture tank 10 has a substantially cylindrical shape as a whole, and a support column (in the culture tank 10 is erected vertically from the center of the bottom surface 17 of the culture tank 10 and fixed to the bottom surface of the culture tank 10 ( (Stirring shaft) 20 and a stirring means 30 rotatably attached to the support column 20 are provided. The support column 20 is arranged so that the central axis 14 thereof is coaxial with the central axis of the culture tank 10.

  The support column 20 has a conical portion 22 formed in a conical shape in which the fixed portion to the bottom surface 17 in the culture tank 10 is expanded in the downward direction, and the liquid flow in the vicinity of the center of the bottom due to the rotation of the culture solution. Itching has no room for it to be occupied by the conical portion 22, and therefore there is no precipitation of cell clumps that increase in specific gravity with cell growth. The support column 20 is not fixed to the culture vessel 10 but may be integrally formed at the time of molding. A stirring means 30 is rotatably attached to the upper portion 24 of the support column 20. In the column 20, the thickness of the portion from the upper part of the conical part 22 to the upper part 24 is not particularly limited, but from the viewpoint of ensuring the strength of the column 20, the lower part is thickened and the diameter is gradually reduced upward. It is preferable that

  The culture tank 10 includes a port 12 that protrudes radially outward from the side wall 11 thereof. The port 12 is provided in the lower part of the culture tank 10 so that the longitudinal axis 13 thereof is perpendicular to the central axis 14 passing through the center of the support 20 in the culture tank 10. The inner end 12a of the port 12 is flush with the inner wall surface of the culture tank 10 (inner wall surface of the side wall 11). In addition, two screw strips 12 c are provided on the outer end portion 12 b of the port 12. The port 12 may be configured as a female luer connector that fits (screws) with a sensor connector described later. The inner peripheral surface 12d of the port 12 is formed in a tapered shape that tapers toward the base end side (left side in FIG. 2A). The outer peripheral surface of the port 12 other than the thread 12c may be formed in a tapered shape or the same diameter as the inner peripheral surface 12d.

The culture tank 10 is also provided with a port 15 that protrudes obliquely upward from the side wall 11 of the culture tank 10 to the upper side of the culture tank 10 in the radial direction, and further, the port of the culture tank 10 around the central axis 14. A port 16 projecting obliquely upward from the side wall 11 radially outward is provided at a position 180 degrees away from 15 and above the port 15. The ports 13, 15, and 16 can be formed integrally when the culture tank 10 is molded, or the ports 13, 15, and 16 can be fixed to the culture tank 10 after being molded separately.

  A culture solution is placed in the culture tank 10. Therefore, the culture tank 10 and the support | pillar 20 are formed with the material which is inert with respect to a culture solution component, does not have cytotoxicity, and has tolerance to sterilization (it is also called decontamination, disinfection, or aseptic) processing. Examples thereof include glass, synthetic resin, and stainless steel. The internal volume and shape of the culture tank 10 are appropriately determined according to the amount of the culture solution. The internal volume (total volume) of the culture tank 10 is not particularly limited, and examples thereof include 20 ml to 1000 ml. From the viewpoint of agitation and aeration efficiency, the shape of the culture tank 10 is such that when a desired amount of culture solution is introduced, the diameter and the liquid depth in the culture vessel become 1: 1, and the location of the culture solution is relative to the internal volume. It is preferable to design the quantification to be about half.

  The stirring means 30 is preferably formed of a material that is inert to the culture medium and has resistance, and examples thereof include synthetic resins and stainless steel. The stirring blade 34 is also preferably formed of a plate material that is inert to the culture medium and has resistance, and examples thereof include a thin plate-like synthetic resin and stainless steel (for example, SUS316 having a thickness of 1 mm). The stirring blade 34 of the stirring means 30 has a magnetic body (permanent magnet or the like) 36 coated with tetrafluoroethylene or the like at its lower end. The magnetic body 36 can be fixed and held by a bent portion of the lower end of the stirring blade 34.

  Here, although two right and left stirring blades 34 are shown in FIG. 3, the present invention is not limited to this. About the number of the stirring blades 34, although it depends on the number of rotations of the stirring means, it is preferable from the balance of the stirring blades 34 at the time of stirring to equip the struts 20 at equal intervals, for example, 2 to 4 blades are suitable. . Further, the number of rotations of the stirring means during cell culture is not particularly limited, and a number of rotations that ensures low shear stress, prevention of precipitation of cell aggregates, and the like is appropriately selected and adopted. 80 rpm is preferred.

  Further, a screw 18 protruding outward from the wall surface 17 is provided above the culture tank 10. This is for fixing a lid (not shown) to the culture tank 10 with screws.

4A is a front view of the sensor connector of the present invention, and FIG. 4B is a cross-sectional view taken along line XX of FIG. 4A. In the present invention, the sensor connector is formed in a cylindrical shape as a whole, which is composed of two sections of a cylindrical distal end side portion and a proximal end side portion. 4, the sensor connector 40 is a male luer connector of the luer connector, the tip side portion of the cylindrical large-diameter portion A and the proximal side is a (left side in FIG. 4) (right side in FIG. 4) It consists of two sections with a certain small diameter part B, and is formed in the stepped cylindrical shape as a whole. In the present invention, as described above, it may be stepped or may be stepless.

  The large-diameter portion A at the distal end side portion of the sensor connector 40 includes an outer cylinder portion 42 and an inner cylinder portion 44. The outer cylinder part 42 and the inner cylinder part 44 are connected to each other on the base end side of the large diameter part A by an inner bottom surface 42b. The outer cylinder portion 42 includes a thread 42 a that engages with the thread 12 c of the port 12 on the inner surface thereof. The outer peripheral surface 44a of the inner cylinder part 44 is formed in the taper shape which tapers toward the front end side. In addition, the inner peripheral surface 44b of the inner cylinder part 44 may be formed in a taper shape similarly to the outer peripheral surface 44a, or may be formed in the same diameter. The outer peripheral surface 44a of the inner cylinder portion 44 is a surface that abuts on the inner peripheral surface 12d of the port 12 that performs the same function as a female luer connector, for example. Since the outer peripheral surface 44a of the inner cylindrical portion 44 is tapered so as to taper toward the tip end side, when the sensor connector 40 is fitted to the port 12, the inner peripheral surface 12d of the port 12 and the outer peripheral surface 44a of the inner cylindrical portion 44. Are fixed in a liquid-tight contact state.

  The length of the inner cylindrical portion 44, that is, the length from the inner bottom surface 42 b of the outer cylindrical portion 42 to the outer side surface 48 a of the distal end wall portion 48 of the inner cylindrical portion 44 is determined by connecting the sensor connector 40 to the port 12 of the culture vessel 10. When fitted, the outer surface 48a is formed so as to be substantially flush with the inner wall surface of the culture tank 10. The length of the inner cylinder portion 44 of the sensor connector 40 may be designed in consideration of the thickness of the fluorescent probe described later. The reason for designing in this way is that if the outer side surface 48a protrudes too much from the inner wall surface of the culture vessel 10, the movement of the stirring blade 34 may be disturbed, or the stirring may be disturbed to prevent uniform stirring. This is because stagnation may occur in the flow of the culture solution unless the inner wall of the culture tank is reached.

  A passage 47 for inserting the optical fiber 46 is formed inside the inner cylindrical portion 44 of the sensor connector 40, and the distal end side (left side in FIG. 4) of the passage 47 is closed by the distal end wall portion 48. An outer surface 48a of the tip wall portion 48 extends in a direction perpendicular to the central axis of the sensor connector 40, and the outer surface 48a has a fluorescent probe 49 that emits fluorescence when irradiated with light, such as a patch-type fluorescent probe. Is fixed. The method for fixing the fluorescent probe to the outer surface 48a is not particularly limited. Examples thereof include a method of attaching a patch and a method of directly applying or baking a fluorescent substance on the outer surface 48a. As described above, the patch-type fluorescent probe has a patch shape, and is, for example, a thin-shaped fluorescent probe having an appropriate shape such as a circle. Such a configuration is preferable because it is easy to handle. Further, as the fluorescent probe 49, not only a fluorescent substance containing a fluorescent substance (fluorescent substance) that responds to the dissolved oxygen concentration in the culture solution, for example, a metal porphyrin complex, but also the pH, temperature, and dissolution of the culture solution. It is also possible to prepare a sensor connector to which a fluorescent probe containing a luminescent substance that responds to carbon dioxide gas or the like is fixed. Therefore, the user selects a fluorescent probe from various types of probes as required, and selects a desired one. It becomes possible to measure characteristics. The fluorescent probe 49 may be one in which a fluorescent material is applied to the patch surface, one in which the patch itself is impregnated, one that is directly applied to the outer surface 48a of the tip wall portion 48, or the like. As various fluorescent probes 49 for measuring these characteristics, known ones are effectively used. Further, for example, when the fluorescent probe 49 is a patch type, the thickness is usually less than about 1 mm, and it is fixed to the outer surface 48a of the tip wall portion 48 with a transparent adhesive (for example, silicon adhesive). Is done. The sensor connector 40 can irradiate the fluorescent probe 49 with light and receive the fluorescent light from the fluorescent probe 49 by a known light emitting and receiving unit (not shown) that alternately repeats light emission and light reception for a short time via the optical fiber 46. In addition, it is preferably formed of a light-transmitting material, but for this purpose, at least the tip wall portion 48 of the sensor connector 40 only needs to be formed of a light-transmitting material. The distance between the outer side surface 48a and the inner side surface 48b of the tip wall 48 (the thickness of the tip wall 48) is the distance between the fluorescent probe 49 and the tip of the optical fiber, and thus the measured fluorescence. In order to affect the strength, it is preferable that the thickness be as thin as possible and less than about 1 mm.

  The small-diameter portion B on the proximal end side of the sensor connector 40 is configured as an optical fiber receiving portion that receives the optical fiber 46, and the optical fiber 46 is attached to the sensor connector 40 so that the optical fiber 46 does not easily fall off in the hollow portion inside thereof. A stopper 50 that can be held inside is fitted. As shown in FIG. 4, the stopper 50 is arranged so that the central axis of the stopper 50 is coaxial with the central axis of the sensor connector 40. The stopper 50 has a passage 51 through which the optical fiber 46 passes, and a lip extending at an angle toward the inside of the stopper 50 and toward the tip side of the stopper 50 at the tip (left side in FIG. 4) of the stopper 50. A portion 52 is provided. The tip of the lip 52 is open, and the opening has an inner diameter smaller than the outer diameter of the optical fiber 46 to be inserted. A plurality of (two in the example of FIG. 4) holes 43 are provided in the small-diameter portion B, and protrusions 54 formed on the outer peripheral surface of the stopper 50 are inserted into the holes 43, and the stopper 50 is connected to the sensor connector 40. The stopper 50 is held so as not to come off. The stopper 50 is preferably formed of an elastic material such as rubber in order to hold the optical fiber 46 so as not to easily move in the lip portion 52.

  Further, the outer surface of the sensor connector 40 has a plurality of wings 45 (2 in the example of FIG. 4) that allow the user to easily rotate the sensor connector when the sensor connector 40 is fitted to the port 12. One) provided.

  As described above, the material of the sensor connector 40 is not particularly limited as long as at least the tip wall portion 48 is formed of a material that is light-transmissive and does not have autofluorescence. For example, a synthetic resin (e.g., polycarbonate, (Polystyrene) and the like are preferable.

As shown in FIG. 3, by rotating the sensor connector 40 having the above structure, when the port 12 of the culture tank 10 at the deepest position of the sensor connector 40 is attached to the sensor connector 40 and port 12, the sensor The central axis of the connector 40 and the central axis of the port 12 are positioned with respect to each other so as to be coaxial. At this time, the outer surface 48 a of the tip wall portion 48 of the sensor connector 40 is substantially flush with the inner wall surface of the culture vessel 10. Therefore, it is possible to suppress the movement of the stirring blade 34 or the occurrence of stagnation in the culture solution. Then, the tapered shape of the sensor connector 40 and the port 12 as described above allows the surfaces to be frictionally engaged with each other, and the position is fixed so that the sensor connector 40 does not protrude or retract from the inner wall surface of the culture vessel 10. The In the state where the position is fixed, the fluorescent probe 49 is formed perpendicular to the central axis of the sensor connector 40 as described above, so that the optical fiber 46 is also positioned so as to be perpendicular to the fluorescent probe 49. ing. Therefore, it becomes easy to make the positional relationship and the angle relationship between the fluorescent probe 49 and the optical fiber 46 appropriate to each other, and the work for attaching the optical fiber 46 and the fluorescent probe 49 to the culture vessel 10 is also much more difficult than before. It becomes a simple thing. Further, the optical fiber 46 is inserted into the sensor connector 40 until the tip of the optical fiber 46 comes into contact with the bottom surface of the passage 47 of the sensor connector 40 (the inner surface 48b of the tip wall portion 48). Is accurately maintained, and reproducibility (that is, the distance and angle between the two are always properly positioned) is improved. Moreover, since the sensor connector 40 is liquid-tightly fixed to the port 12 by the taper shape, leakage of the culture medium from the port 12 is prevented.

  In addition, the port 12 can be attached to containers of various sizes and shapes. For example, the same sensor connector 40 can be used by providing the port 12 having a common luer taper in culture vessels of various volumes. Therefore, it is possible to achieve a significant cost reduction.

  In addition, in the existing culture tank, the sensor connector 40 of the present invention is provided by providing a flow cell having a port having the same luer taper as the port 12 common to the line for circulating the culture solution outside the culture tank. Can be used.

  Furthermore, the same luer taper as that of the port 12 described above can be attached to an appropriate position of a known culture bag, and the sensor connector 40 can be used in the same manner.

  Furthermore, as described above, not only a fluorescent probe containing a fluorescent substance that emits fluorescence in response to the dissolved oxygen concentration in the culture solution, but also a fluorescent probe containing a fluorescent substance that responds to the pH of the culture solution, etc. were fixed. It is also possible to prepare a sensor connector. Therefore, the user can select a fluorescent probe from various types of fluorescent probes as necessary, and perform measurement of desired characteristics. Needless to say, the number of ports 12 is not limited to one as in the above-described embodiment, and a plurality of ports 12 may be provided according to the number of characteristics to be measured.

  The culture apparatus of the present invention can be disposable.

DESCRIPTION OF SYMBOLS 10 Culture tank 11 Side wall 12 Port 12a Inner edge part 12b Outer edge part 12c Thread 12d Inner peripheral surface 40 Sensor connector A Large diameter part B Small diameter part 42 Outer cylinder part 42a Thread 43 Hole 44 Inner cylinder part 44a Outer peripheral surface 44b In Peripheral surface 46 Optical vibrator 48 Tip wall 48a Outer side 48b Inner side 49 Fluorescent probe 50 Stopper 51 Passage 52 Lip part 54

Claims (5)

A culture device in which a measuring means for measuring a predetermined characteristic of a culture solution is detachably connected to a culture tank via an optical fiber,
The culture tank has a substantially cylindrical shape as a whole,
The culture tank includes a generally cylindrical port extending radially outward from a side wall of the culture tank, and the port has a longitudinal axis of the port with respect to a central axis of the substantially cylindrical culture tank. Are arranged so as to be vertical,
The culture apparatus has a fiber optic receiving portion that removably receives the optical fiber of the measuring instrument, and is a sensor connector that can be fitted to the port. The sensor connector has a cylindrical distal end side and a proximal end side. A sensor connector having a cylindrical shape as a whole having two sections with a portion, wherein the tip side portion includes an outer cylindrical portion on the outside and an inner cylindrical portion disposed on the inner side of the outer cylindrical portion. Prepared,
The port has an inner end on the side wall side of the culture tank, an outer end opposite to the inner end, and an inner peripheral surface that defines a passage for receiving the sensor connector. A thread projecting outward in the radial direction is formed in a portion adjacent to the outer end of the port, and the inner peripheral surface of the port tapers from the outer end toward the inner end. Is formed as
The outer peripheral surface of the inner cylinder part of the sensor connector is formed to taper toward the tip of the inner cylinder part,
On the inner peripheral surface of the outer tube portion of the sensor connector, a thread that can be threadedly engaged with the thread of the port is formed,
The tip of the inner tube portion of the sensor connector is closed by a tip wall portion, the tip wall portion is perpendicular to the central axis of the sensor connector, and the optical fiber is disposed on the outer surface of the tip wall portion. A fluorescent probe containing a fluorescent substance that emits fluorescence corresponding to a predetermined characteristic of the culture solution by light irradiated through is fixed.
A culture apparatus, wherein a stopper for holding an optical fiber in the sensor connector is provided in a portion of the sensor connector farther from the culture tank than the inner cylinder part and the outer cylinder part.   The length of the inner cylinder part of the sensor connector is such that the tip wall part of the inner cylinder part is flush with the inner surface of the side wall of the culture tank when the sensor connector is attached to the port. The culture apparatus according to claim 1.   The culture apparatus according to claim 1 or 2, wherein the fluorescent probe fixed to the outer surface of the tip wall portion of the sensor connector has a patch shape.   The culture apparatus according to claim 1, wherein the port is formed as a female luer connector, and the sensor connector is formed as a male luer connector. A culture apparatus according to any one of claims 1 to 4, and a light emitting / receiving section for irradiating the fluorescent probe of the culture apparatus with light through an optical fiber and receiving fluorescence emitted by the fluorescent probe. A culture device system comprising a measuring means.

Images