The present invention relates to an automated culture device for culturing a cell or a tissue.
Regenerative medicine for recovering the function of an internal organ by using a biological sample such as a regenerated tissue produced from a cell as a raw material is expected as a curative treating method for diseases which have had no treatments. Regenerative tissues are produced based on SOP (Standard Operational Procedure) by a person engaged in manufacture and having a professional cell culturing technology at a CPC (Cell Processing Center) which provides a clean manufacturing environment. Therefore, the production of the regenerated tissues requires huge amounts of labor costs, time and operational cost. Since all the production steps are performed manually, there is limitation to the amount of a regenerated tissue to be produced. Therefore, production costs become high, thereby preventing the spread of regenerative medical treatments.
To break through this current situation, the introduction of an automated culture device which automates part or all of the culturing step is desired. By carrying out the culturing step by means of an automated culture device and not manually, labor saving and cost reduction can be realized, thereby making mass-production possible. In addition, since the operation of the automated culture device is constant, it is expected that this contributes to the constant quality of a regenerated tissue obtained after production.
As background art in this technical field, there is, for example, Patent Literature 1. This literature discloses that, in a culture device having a plurality of culture chambers, to solve a problem that it is troublesome to store and take out an object to be cultured into and from a culture chamber as compared with a culture device having only one culture chamber, a pin having a connector (female) is connected to the ground so that when a control unit detects that the pin is connected to the ground at the time of mating a connector (male) so as to detect that the pin is situated at the mating position, the control unit supplies a gas, power and a signal to a culture chamber and when the control unit detects that the pin is not at the mating position, the control unit stops the supply of a gas, power and a signal.
PTL 1: Japanese Patent Application Laid-Open No. 2006-149232
For the quality inspection of a cultured cell, there is SOP in which some of culture vessels are selected at random and cells are taken out from the culture vessels to be inspected. Since quality inspection includes a time-consuming step, culture vessels are taken out, for example, one day before the end of the culturing period determined by SOP. In this case, the automated culture device continues to carry out medium or gas exchange for culture vessels left after the removal of the above culture vessels. However, medium or gas exchange should not be carried out in the routes of the removed culture vessels. This is because the medium or the gas may leak into the device or may remain in tubes connecting a medium supplier to the culture vessels as the destinations of the medium and the gas are not existent.
In Patent Literature 1 described above, although gas supply to the removed culture vessel is stopped, the gas remains in a flow channel. Although Patent Literature 1 is silent about medium exchange, when it is carried out with the same constitution, it is considered that the medium remains in the flow path. When medium exchange is carried out for another culture vessel while the medium remains in the flow channel, this amount of the medium in the flow channel flows into the culture vessel, in addition to the amount of a medium to be exchanged, whereby the medium overflows from the culture vessel, thereby making it impossible to continue culture.
It is an object of the present invention to provide an automated culture device which solves the above problem and does not perform medium exchange or gas exchange for the removed culture vessel.
To attain the above object, the present invention provides an automated culture device which comprises a plurality of culture vessels, flow channels connected to the respective culture vessels, a vessel selection unit for controlling a liquid or gas to be supplied to the culture vessels through the flow channels, and a control unit for controlling the vessel selection unit, wherein the control unit detects the disconnection of flow channels corresponding to some of the culture vessels to change the control of liquid supply or gas supply.
According to the present invention, since medium exchange or gas exchange is not carried out for the flow channels of the removed culture mediums, the medium or the gas does not leak into the device or does not remain in the flow channels.
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.
The vessel selection unit 300 for supplying a liquid or gas switches between the supply of a liquid and the supply of a gas based on the control of the control unit 400 and selects a culture vessel to be operated. Details of this constitution will be described hereinafter. The culture vessel 500 is sealed except for tubes 501, 502 and 503 connected thereto and stores a cell filled in a medium. The culture vessels 510, 520 and 530 have the same structure as the culture vessel 500. The culture supernatant collection selection unit 900 selects a culture vessel from which a culture supernatant is to be collected based on the control of the control unit 400. Details of this constitution will be described hereinafter. A culture supernatant bottle 600 stores a culture supernatant discharged from the culture vessels at the time of medium exchange.
An air filter 610 discharges the inside air to the outside therethrough in order to prevent the inside pressure of the culture supernatant bottle 600 from becoming a positive value when a culture supernatant flows into the culture supernatant bottle 600 at the time of medium exchange. An air filter 700 discharges a gas from a culture vessel to the outside at the time of gas exchange. A filter having a pore diameter of, for example, 0.22 μm, is used as the air filters 210, 610 and 700 in order to prevent the contamination of the inside of a culture vessel by bacteria contained in the outside air. The flow channels of the medium and the gas of the automated culture device are closed by this constitution.
The constitution of the vessel selection unit 300 for supplying a liquid or gas in this example will be described with reference to
The constitution of the culture supernatant collection selection unit 900 of this example will be described with reference to
The control unit 400 carries out medium exchange or gas exchange based on a culture schedule input by a person engaged in manufacture according to SOP at the start of cell culture. The culture schedule includes the times of starting medium exchange and gas exchange from the start to the end of cell culture. This control unit 400 can be constituted by using an ordinary computer which includes a controller, a central processing unit (CPU) for running programs, a memory for storing programs and data, and input and output units such as a display and a mouse.
The gas exchange operation of the automated culture device of this example will be described with reference to
A description is subsequently given of the medium exchange operation of the automated culture device of this example with reference to
After the end of the movement of the culture supernatant in the culture vessel 500, the medium in the medium bottle 200 is supplied into the culture vessel 500. The control unit 400 instructs the vessel selection valve 340 to open the valve 341. Then, the control unit 400 instructs the liquid/gas supply selection valve 320 to open the liquid supply valve 322. Subsequently, the control unit 400 instructs the operation of the pump 310. The medium sucked out from the medium bottle 200 is supplied into the culture vessel 500 through the liquid supply valve 322, the valve 341 and the tube 501. After a predetermined time during which the amount of the medium determined by SOP is supplied, the control unit 400 stops the operation of the pump 310 and instructs the liquid/gas supply selection valve 320 to close the liquid supply valve 322 and the vessel selection valve 340 to close the valve 341.
According to SOP, the culture vessel is generally taken out one day before the end of the culturing period. As for the operation of taking out the culture vessel, two positions of each of the tubes connected to the culture vessel are heat-welded to be closed, and a part between the two positions is cut out to maintain the closing properties of a flow channel.
Operation from the time of starting the gas exchange of the culture vessel 510 specified in the culture schedule in the automated culture device in the state shown in
When the inside pressure of the flow channel is lower than a threshold value close to the predetermined inside pressure of the cylinder 100, for example, 0.05 MPa, medium exchange is carried out in the same manner as medium exchange for the culture vessel 500 described previously at the time of starting medium exchange after the time of 5100 specified in the culture schedule. When the inside pressure of the flow channel is not lower than the threshold value close to the predetermined inside pressure of the cylinder 100, the control unit 400 detects that the culture vessel 510 has been removed. When the control unit 400 detects that the culture vessel 510 has been removed, the gas in the flow channel is discharged (S130). The gas is discharged by opening the exhaust valve 350 after the gas supply valve 321 and the valve 342 are closed. The supply of the medium is not carried out without opening the liquid supply valve 322 at the time of medium exchange after the time of 5100 specified in the culture schedule.
Thus, the control unit 400 of the automated culture device of this example has means of detecting the removed culture vessel so that it can perform the control of not carrying out medium exchange for the removed vessel. According to the constitution of this example, the medium does not remain in the flow channel, thereby eliminating the risk of overflowing the medium from the culture vessel.
As described above, in the constitution of the automated culture device of this example, the detection of the removed culture medium is carried out by the detection of the inside pressure of the flow channel by means of the pressure sensor 330, and the control unit 400 judges whether medium exchange is necessary or not. Since only a required amount of the medium is used in the constitution of this example, the volume of the medium stored in the medium bottle can be reduced, thereby making it possible to reduce the size of the medium bottle and also the size of the device. Further, since the amount of the expensive medium in use can be reduced, running cost for each time of cell culture can be cut.
In the automated culture device of this example, a cell in the culture vessel is observed through a microscope not shown in
Although culture exchange was not carried out without opening the liquid supply valve 322 in Example 1 described above, the control unit 400 may control such that medium supply is not carried out without operating the pump 310 at the time of starting medium exchange specified in the culture schedule when it detects the removed culture vessel. When the medium is not supplied without opening the liquid supply valve 322, the medium slightly moves in the flow channel from the medium bottle 200 to the liquid supply valve 322. Although the medium moved into this flow channel may flow into an unexpected place at the time of the medium exchange of another culture vessel, since the medium is not supplied from the medium bottle 200 without operating the pump 310, the amount of the medium remaining in the flow channel can be made nil.
As a second example, a description is subsequently given of an automated culture device which detects the removal of a culture vessel from a photographed image of a microscope.
The vessel selection unit 301 for supplying a liquid or gas of the automated culture device shown in
A method of judging whether a culture vessel is existent or not from a photographed image of the microscope by means of the automated culture device of this example will be explained with reference to the flow chart of
As a result of the detection of the existence or nonexistence of the culture vessel, when the culture vessel exists, medium exchange is carried out at the time of starting medium exchange specified in the culture schedule. Medium exchange is carried out in the same manner as medium exchange for the culture vessel 500 described in Example 1. When the culture vessel does not exist, culture exchange is not carried out at the time of starting medium exchange specified in the culture schedule after the time of S200.
That is, the control unit 400 of this example has means of detecting the removed culture vessel and can control such that culture exchange is not carried out for the removed vessel. In this example, the medium does not remain in the flow channel, thereby eliminating the risk of overflowing the medium from the culture vessel like Example 1. Since only a required amount of the medium is used in this example, the volume of the medium stored in the medium bottle can be reduced, thereby making it possible to reduce the size of the medium bottle and also the size of the device. Further, since the amount of the expensive medium in use can be reduced, running cost for each time of cell culture can be cut.
Further, whether a culture vessel is existent or not can be judged from the result of a photographed image of the microscope, and gas exchange is not carried out for the removed vessel in this example. When gas exchange is carried out for the removed culture vessel, the gas remains in the flow channel. The remaining gas continues to apply excessive pressure to the end of the closed tube. If closing properties are lost by the broken tube, biochemical contamination may occur. Since a gas pressure change in the flow channel can be made small in this example, it is easy to maintain closing properties. It is also easy to design the strength of the tube and the pressure resistance performance of the tube at the time of disconnection.
When gas exchange is carried out for the route of the removed culture vessel, the inside pressure of the flow channel rises, whereby a big pressure change occurs at the time of the gas exchange of another culture vessel and becomes stress to a cell, thereby affecting the result of culture. Since a pressure change at the time of starting gas exchange is small in the constitution of this example, stress to a cell can be reduced and quality can be improved.
When gas exchange is carried out for the route of the removed culture vessel, the inside pressure of the flow channel rises, whereby it is erroneously detected that gas exchange fails for some reason. The processing of checking the gas pressure can be simplified and the detection accuracy of the gas pressure is enhanced in this example.
In the above explanation, an example in which a phase microscope is used as the microscope 800 of this example has been described. As described previously, the phase microscope is often mounted in an automated culture device to observe a transparent cell. The detection of a culture vessel is carried out by means of a microscope for cell observation, thereby making it possible to reduce the size of the device. Further, the cost of the device can be reduced.
Since a photographed image of the phase microscope is expressed only by a luminance value, it is difficult to detect color. The existence of a culture vessel may be detected by detecting the color of a medium by means of a stereomicroscope as the microscope 800 and checking whether the detected color falls within the range of changed color of phenolphthalein contained in an ordinary medium. To detect color, there are a method of detecting color from the RGB values of an image and a method of detecting color from HSV (Hue, Saturation, Value). Since color detection is easier than cell detection, the processing of detecting the existence of a culture vessel can be simplified and the detection accuracy can be improved. Further, the detection processing can be accelerated.
While the method of detecting a cell for the detection of a culture vessel has been described in this example, the end of a culture vessel may be detected by moving the microscope 800 having a camera function or a photographing unit such as a camera to the end of the culture vessel to carry out high-pass filter image processing on a photographed image for edge detection. This is because edge detection can be easily performed by high-pass filter processing when using an image of the end of a culture vessel whose flow channel has been disconnected at the end of the culture vessel connected to the flow channel such as a tube. Since high-pass filter processing has a small operation amount and is easier than cell detection, the detection of the existence of a culture vessel can be simplified and the detection accuracy can be improved. Further, the detection processing can be accelerated.
As means of detecting the existence of a culture vessel, a reader unit such as a bar code reader for reading a matrix type two-dimensional barcode code typified by a QR code (registered trademark) or a marker pattern such as bar code may be used. Which culture vessel has been removed can be detected and the detection accuracy can be improved by adding reference codes for distinguishing culture vessels 510 to 530 to the marker pattern. Further, the detection processing can be accelerated.
As a third example, an automated culture device in which a person engaged in manufacture who has removed a culture vessel inputs information on the removed culture vessel into the control unit will be described.
The input unit 410 of the automated culture device of
In the automated culture device of this example, the number of the removed culture vessel can be transmitted to the control unit 400 without being erroneously detected by the removal operation of a person engaged in manufacture and the explicit input of the number while he/she holds the removed culture vessel by hand, in addition to the effects shown in Example 1 and Example 2.
The number of the removed culture vessel may be input by causing the input unit 410 to read a marker pattern attached to the culture vessel.
The number of the removed culture vessel may be input by attaching an electronic tag to the culture vessel to be read by the input unit 410. In this case, the input screen shown in
Input into the input unit 410 may be made automatically and not manually by a person engaged in manufacture. For example, when an unshown contact sensor is installed on a floor for installing culture vessels and a culture vessel is removed by a person engaged in manufacture, the control unit 400 detects from the contact sensor that the culture vessel has been removed. In this case, the contact sensor functions as the input unit 410. According to this constitution, it is possible to prevent a person engaged in manufacture from forgetting the input of the number of the removed culture vessel.
Further, a detection sensor which detects the separation of a connector which is connected to a tube connected to a culture vessel when the culture vessel is removed may be installed to input the number of the culture vessel into the control unit 400. In this case, the detection sensor functions as the input unit 410. Since a person engaged in manufacture can remove the culture vessel while it is separated from the floor surface, working efficiency is improved.
Further, the input unit 410 may be installed in the opening section of an incubator storing the automated culture device so that an electronic tag is detected when a culture vessel leaves the incubator. Since the operation of removing the culture vessel is completed by taking out the culture vessel from the incubator, the timing of detecting the removed culture vessel can be made more accurate.
The detection of the removal of a culture vessel from the inside pressure of the flow channel shown in the first example may be combined with the input of the removed culture vessel shown in the third example. In
While preferred examples of the present invention have been described with reference to the accompanying drawings, it is needless to say that the present invention is not limited thereto. It is obvious that a person skilled in the art can arrive at variations and modifications within the scope of a technical idea described in claims, and it is to be understood that they fall within the technical scope of the present invention as a matter of course.
For example, the above examples have been described in detail to make it easy to understand the present invention and not limited to those having all the constitutions explained above. Part of the constitution of a certain example may be replaced by the constitution of another example, and the constitution of a certain example may be added to the constitution of another example.
Further, while an example in which a computer program is prepared to realize part or all of the above functions or the control unit has been described, it is needless to say that part or all of the functions or the control unit may be realized with hardware by designing with an integrated circuit.
100: cylinder
200: medium bottle
210: air filter
300: vessel selection unit
310: pump
320: liquid/gas supply selection valve
330: pressure sensor
340: vessel selection valve
350: exhaust valve
360: air filter
400: control unit
410: input section
500: culture vessel
600: culture supernatant bottle
610: air filter
700: air filter
800: microscope
810: microscope slider
900: culture supernatant collection selection section
910: vessel selection valve
950: pump
Filing Document | Filing Date | Country | Kind |
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PCT/JP2013/078857 | 10/24/2013 | WO | 00 |