JP2670326B2 - Culture solution collection device, collection method and culture system - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method and an apparatus for collecting a culture solution from a culture tank for culturing cells of an organism, and is particularly necessary for monitoring and controlling the culture condition. The present invention relates to a sampling method and a sampling device suitable for collecting a small amount of a sample without causing microbial contamination, and a culture system including the sampling device.

[Conventional technology]

Various devices have been devised as a device for collecting a culture solution in a conventional culture tank, and it is widely practiced to collect a sample while preventing contamination of various bacteria. FIG. 3 shows an example of the concept of a known sampling mechanism that is normally used.
In each case, the infiltration of bacteria into the culture tank is prevented by sterilizing the inside of the pipe for collection with a steam line after collecting the sample.

[Problems to be solved by the invention]

The following points should be noted when collecting the culture solution. That is, it is necessary to extract the culture solution at a flow rate at which the cells in the culture solution do not settle in the sampling tube. In addition, the linear velocity that gives strong turbulence is required to resuspend the cells that were retained in the tube at the previous collection and deposited on the tube wall. For monitoring, a sample taken after the cells deposited in the tube have been removed must be used.

In the above conventional method, directly through the steam in the pipe,
It is necessary to maintain a high temperature of 121 ° C or higher for a certain period of time, and the pipe diameter will be determined by the required steam flow rate. Therefore, the inner diameter of the sampling pipe changes depending on the pipe length, the presence or absence of heat insulating material, etc.
It becomes about 10mm. As a result, it becomes meaningless as a monitoring sample of the culture solution unless it is collected at least 100 ml / time.

In recent years, various measurement techniques have advanced, and glucose, lactic acid,
The amount of sample used for quantification of various amino acids and various physiologically active substances is becoming small. In the conventional method, in order to obtain a sample of several ml, a large amount of culture solution is collected,
Most of it will be discarded. Due to the development of culture technology in recent years, a method of increasing the production efficiency of a target substance by culturing the target cells at a higher density in a smaller culture tank is often used. It has been desired to improve the collection of a large amount of liquid as soon as possible.

It is an object of the present invention to provide a method and apparatus for continuously collecting a small amount of sample.

[Means for solving the problem]

 The above object is achieved by the following.

That is, the present invention is such that one end of the culture solution collection tube having a small inner diameter is projected from the tip of the steam flow tube, the other end of the culture solution collection tube is larger than the outer diameter of the collection tube, and steam sterilization is performed. In the culture medium collection device that is housed in a steam flow pipe having an inner diameter that gives a sufficient cross-sectional area to pass a steam flow rate that can raise the temperature to a necessary temperature,
Further, the steam distribution tube is connected to a culture vessel by a tube material a, a tube material b which is joined by a partition shared with the tube material and has a large inner diameter portion in the vicinity of the partition material, and a tube material b is connected by a large inner diameter portion. In the culture medium collecting device, the culture medium collecting pipe is constituted by the pipe material c, and the culture medium collecting pipe penetrates the partition wall between the pipe materials a and b and has one end opened inside the pipe material.

Further, the present invention, the culture medium collecting device is attached to a culture tank, after the steam is circulated in the culture medium collecting device to sterilize the inside of the collecting device, a pipe material connected to a pipe material having a large inner diameter Is removed and then a sample is collected through a sample collection pipe housed in the tubular material having a large inner diameter.

In this case, the culture solution can be collected by connecting the sample collecting pipe to a flexible pipe connected to a measuring device such as an automatic diagnostic device or an automatic analyzer.

Further, the present invention is a culture tank equipped with a means for supplying gas to culture cells, a means for supplying a culture solution, and a means for discharging a waste culture solution, and one end of a culture solution collection tube having a small inner diameter is projected from the tip of a steam distribution tube. The inner diameter of the other end of the culture solution collection tube, which is larger than the outer diameter of the collection tube, and which provides a sufficient cross-sectional area for passing the steam flow rate capable of raising the temperature to the temperature required for steam sterilization. A culture solution collection device housed in a steam distribution tube having the means, means for enlarging an image of cells in the culture solution collected by the culture solution collection device, and image recognition for recognizing live cells and dead cells from the image A continuous automatic diagnostic device for the number and activity of cells equipped with the device,
Glucose in the culture solution collected by the culture solution collecting device, lactic acid, based on the information from the dissolved component automatic analyzer for analyzing dissolved components such as ammonia, the continuous automatic diagnostic device and the dissolved component automatic analyzer The culture system has means for automatically operating the culture tank.

[Action]

The sampling tube usually has an inner diameter of 5 mm or less, preferably 3 to 1 mm, and its tip is inserted into the culture tank. The portion of the sample collecting tube located outside the culture tank is housed in a steam distribution tube having a diameter larger than the outer diameter of the sample collecting tube. The end of the steam distribution tube on the tip side of the sampling tube has a structure that can be connected to the main body of the culture tank by screwing or another method. The other end of the steam flow pipe has a sufficient volume to accommodate the rear end of the sampling pipe therein and a flexible pipe connected to the sampling pipe. When starting the culture, first, when the culture tank is sterilized, the internal temperature is set to 121 ° C. or higher through steam through the steam distribution pipe connected to the culture tank, and the temperature is maintained for about 20 to 30 minutes. During this time,
Sterilization of the sampling pipe and the flexible pipe connected thereto is performed. After sterilization, inside the steam distribution pipe,
The flexible pipe is pulled out under an aseptic atmosphere and connected to the flexible pipe connected to various measuring devices which have been separately sterilized. The sample is collected by installing a peristaltic pump in the middle of the flexible pipe and operating the peristaltic pump to collect the culture solution. The method for collecting is not particularly limited to the above, and a method in which the culture tank main body is pressurized to push it out due to a pressure difference can be used.

In the culture medium collecting device, the steam flow pipe is joined by a member a having a mechanism for connecting to the culture tank, and a partition shared with the member a, and has a large inner diameter portion near the partition. It is composed of a tubular material b and a tubular material c connected to the tubular material b at a portion having a large inner diameter, and has a structure in which the tip of the culture solution collecting tube is projected from the tubular material a and the rear end is located inside the tubular material c. ing. After the sterilization is completed, the pipe material c is removed, the flexible pipe connected to the culture solution collecting pipe is taken out from the inside, and is connected to the flexible pipe for connecting to various separately sterilized measuring instruments.

The method of collecting the culture solution by this culture solution collecting device is performed as follows. First, a culture solution collecting device is attached to a culture tank.
Steam is passed through the steam flow pipe of this culture medium collecting apparatus to keep the internal temperature at 121 ° C or higher and kept for about 20 to 30 minutes. During this period, the sample collection tube and the flexible piping connected thereto are sterilized. After the sterilization is completed, the flexible pipe is pulled out from the inside of the steam distribution pipe in an aseptic atmosphere and connected to the separately sterilized flexible pipe connected to various measuring instruments and the like. A peristaltic pump is installed in the middle of the flexible pipe, and the peristaltic pump is operated to collect the culture solution. By this culture solution collection method, it is possible to continuously and reliably collect a small amount of culture solution aseptically without contaminating the culture tank.

This culture medium collection device is a culture tank, a continuous automatic diagnostic device for the number and activity of cells, an automatic analyzer for dissolved components in the culture liquid,
A culture system that is incorporated into a culture system consisting of a computer for automatically operating a culture tank based on information from the continuous automatic diagnostic device and the dissolved component automatic analyzer, and has a high control accuracy without the risk of contamination by various bacteria. Can be built.

Hereinafter, the present invention will be described in detail with reference to examples. However,
The present invention is not limited to this embodiment.

Embodiment 1 An embodiment of the present invention will be described with reference to FIGS. 1 and 2.
Incidentally, FIG. 1 is a perspective view of the culture medium collecting device, and FIG. 2 is a sectional view thereof.

The sampling tube 1 usually has an inner diameter of 5 mm or less, preferably 3 to 1 mm
One is inserted into the culture tank 10. The portion of the sampling tube located outside the culture tank is accommodated in a steam distribution tube 2a, 2b, 2c having a cross-sectional area larger than the sampling tube outer diameter and sufficient for steam line sterilization. . The steam distribution pipes 2a and 2b are connected by a common partition wall 3.

The steam flow pipe 2a, which accommodates the terminal side of the sampling pipe inserted into the culture tank, is connected to the culture tank main body by screwing or another method. The inner diameter of the steam flow pipe 2b is partially enlarged in the vicinity of the partition wall, and the steam flow pipe 2c is connected in the enlarged portion. The two are usually connected by screwing or other connection method. The above-mentioned sample collection pipe penetrates the partition wall between the steam distribution pipes 2a and 2b and opens in the enlarged portion of the steam distribution pipe 2d. Further, a flexible pipe 6 such as a silicone rubber pipe or a pipe made of polytetrafluoroethylene which is resistant to steam sterilization is connected to the end thereof, and the flexible pipe is housed in the enlarged portion. Also, it is desirable that the inner diameter of the flexible tube be as close as possible to the inner diameter of the sampling tube.

Next, a method of using the culture solution collecting device will be described with reference to FIG.

Sterilization of the culture solution collecting device is performed by the following procedure. First, the flexible pipe 8 connects between one ends of the steam distribution pipe 2a and the steam distribution pipe 2b. Also steam distribution pipe
Connect flexible tube 9 to one end of 2c. A valve for adjusting the steam flow rate is provided at the end of the flexible pipe 9 (not shown). At the time of steam sterilization of the culture tank 10, the valve provided in the flexible pipe 9 is opened, and steam is passed through the steam distribution pipe 2a, the flexible pipe 8, the steam distribution pipe 2b, and the steam discharge pipe 2c. The internal temperature is
Adjust the opening of the valve to at least 121 ° C. About 20 at 121 ° C
Hold for 3030 minutes (FIG. 5 (a)). By this operation, the sample collecting tube 1 and the flexible tube 6 are sterilized at the same time. After the sterilization is completed, close the valve provided on the flexible pipe 9,
Further, the flexible pipe 8 is also closed in the middle by means such as a pinch cock. After cooling, the steam flow pipe 2c is removed (Fig. 5 (b)). Steam distribution pipe 2b
The flexible pipe 6 housed inside is taken out and connected to the flexible pipe 27 for connecting the measuring end (not shown) to the measuring means under an aseptic atmosphere such as a clean bench (Fig. 5 (c) and (D)). In addition, in order to facilitate the connection with the flexible pipe 6, it is preferable to connect a connecting pipe member 26 having elasticity to the tip of the flexible pipe 27. Flexible pipe 27 and connecting pipe material 26
It is needless to say that sterilization treatment is indispensable in advance by sterilizing means such as an autoclave.

When collecting the culture solution, when the inside of the culture tank is in a pressurized state as compared with the outside, a method of flowing out by a pressure difference is also used, but in general, the flexible tube 6 or 27 is used.
The method of attaching the peristaltic pump to and pulling it out is used.

Embodiment 2 FIGS. 3 (a) and 3 (b) show another embodiment of the present invention, in which (a) is a perspective view and (b) is a sectional view thereof.

The front end of the sampling tube is inserted into the culture tank, and the rear end is
Steam distribution pipe having a diameter larger than the outer diameter of the sampling pipe
It is housed in 2a and 2c. The steam flow tube 2a that accommodates the tip side of the sample collection tube 1 is connected to the wall of the culture tank by screwing or another method. The inner diameter of the rear end of the flow pipe 2a is partially enlarged, and the steam flow pipe is
Connected with 2c. The rear end of the sampling tube is a steam distribution tube 2c
There is an opening in the enlarged part of. Furthermore, a flexible tube 6 such as a silicone rubber tube or a polytetrafluoroethylene official is connected to the end, and the flexible tube is stored in the enlarged portion.

The method of use of this embodiment is almost the same as that of the first embodiment.

Embodiment 3 FIGS. 4 (a) and 4 (b) show another embodiment, in which (a) is a perspective view thereof and (b) is a sectional view thereof.

The front end of the sampling tube 1 is inserted into the culture tank, and the rear end is
Steam distribution pipe having a diameter larger than the outer diameter of the sampling pipe
It is housed in 2c and 2b. The steam flow pipe 2b is provided with a connecting means by screwing or other means for connecting to the culture tank, the sample collecting tube penetrates the connecting means, and its tip is in the culture tank. Is inserted. The steam distribution pipes 2c, 2b are connected by a connecting means such as screwing, and the two can be separated as necessary. The rear end of the sampling pipe is opened into the space formed by the steam flow pipes 2b and 2c, and the flexible pipe 6 such as a silicone rubber pipe or a polytetrafluoroethylene pipe is connected to the end of the space. It is housed inside.

Before using this embodiment, first connect the steam supply pipes and the steam drain pipes to the respective end portions 5 and 7 of the steam distribution pipes 2b and 2c, and then connect the steam distribution pipes.
Pass steam through 2b and 2c and adjust the flow rate so that the internal temperature is 121 ° C or higher. The sample collection tube and the flexible tube 6 are sterilized by holding at 121 ° C. for 20 to 30 minutes. After the sterilization is finished, the distribution of steam is stopped, and after cooling, it is used in the same manner as in the above-mentioned embodiment.

Example 4 FIG. 7 shows an example of a culture system using the culture solution collecting device according to the present invention.

Heat-sterilized stainless steel culture tank 10
Culturing animal cells and microorganisms. Although this example shows an example of animal cells, the same applies to the case of microorganisms. The culture tank 10 contains a suspension 11 containing animal cells. Animal cells are cells collected from an organ (eg, liver) of an animal (eg, rat). The useful substance produced by the animal cells is recovered by the culture. Substrates include glucose as an energy source as well as amino acids and serum. If an appropriate environment is maintained, animal cells ingest oxygen and substrates and grow by cell division. The suspension 11 in the culture tank 10 is gently stirred by a stirring motor 12 and a stirring blade 13 to the extent that cells are not damaged, and at the same time, a compressor 14 and a bubble generator.
15 to blow oxygen-containing gas. The rotation speed of the agitating motor 12 and the gas supply amounts of the compressor 14, oxygen 1, and carbon dioxide 2 are controlled by the computer 100 and appropriately operated. The physicochemical properties of the suspension 11 containing the substrate and animal cells are measured by various sensors. That is, the temperature sensor 16, the pH sensor 17, the DO sensor 18, and the DCO ₂ sensor 19 measure the temperature, pH, DO (dissolved oxygen concentration) and DCO ₂ (dissolved carbon gas concentration) of the suspension, respectively. These measurement signals are transmitted to the inference mechanism 95.

The culture solution is collected from the culture tank by the collecting device 20. That is, the sampling pump 22 is driven by a command from the computer 100 to transport the suspension 11 to the mixer 23. The liquid in the mixer 23 is transported to the flow cell 41. The dyeing liquid supply pump 32 is driven in response to a command from the computer 100, the dyeing liquid 31 is transported to the mixer 23, and the suspension 11 and the dyeing liquid 31 are mixed. In response to a command from the computer 100, the transport pump 35 transports the mixed liquid of the suspension 11 and the staining liquid 31 to the flow cell 41. Further, in order to regularly clean the flow cell 41, the cleaning liquid supply pump 34 is driven according to a command from the computer 100, and the cleaning liquid 33 is injected immediately before the flow cell 41. Since the frequency of the cleaning operation is low, it may be executed manually.

In the flow cell 41, an image of cells in the liquid is magnified by an image magnifying device 42 such as a microscope. The image obtained by the image enlarging device 42 cannot grasp the accurate cell state with only one screen, so
The captured images are updated one after another. For this purpose, the XY stage 43, which is an image updating device, is driven in response to a command from the computer 100 to move the flow cell 41 in the X and Y directions. In this way, by moving the flow cell 41, the image enlarging device 42 sequentially obtains images of different portions of the flow cell 41.
This image is captured by the television camera 44, and the signal of the captured image is transmitted to the A / D converter 51.

The A / D converter 51 converts the analog signal of the image into a digital signal. The A / D converted image signal is stored in the image memory 51M. The multivalued image stored in the image memory 51M is transmitted to the live cell image recognition device 60 and the dead cell image recognition device 70. The live cell image recognition device 60 recognizes all cells regardless of whether they are alive or dead, and calculates the number, size, shape, brightness of cells, and the like. The dead cell image recognition device 70 recognizes all cells in a low activity state including a dead state, and similarly calculates the number, size, shape, brightness of cells and the like.
Each calculation result is transmitted to the activity diagnostic device 80. The activity diagnostic device 80 receives the results of these calculations and calculates the number, size, shape, and brightness of the living cells and dead cells, and at the same time, comprehensively diagnoses the active state of the cell group from these calculation results.

The sample suspension is transferred to the flow path switching valve 24 to the automatic analyzer 55. The automatic analyzer 55 automatically measures components dissolved in a liquid such as glucose, lactic acid, and ammonia. The obtained measurement signal is transmitted to the inference mechanism 95.

The knowledge base 90 stores experimentally or theoretically organized knowledge about cell proliferation and metabolism. Reasoning mechanism 95
Is the temperature sensor 16, pH sensor 17, DO sensor 18 and D described above.
The measurement value of the CO ₂ sensor 19 and the diagnostic result of the activity diagnostic device 80,
In addition, the state of the cell is inferred based on the knowledge (rules) of the knowledge base 90 to instruct an appropriate control policy. This instruction result is transmitted to the computer 100. The computer 100 controls oxygen supply, stirring, and substrate exchange based on the instruction result.

This system can greatly improve the control accuracy of culture.

〔The invention's effect〕

According to the present invention, it is possible to use a sampling tube having a small inner diameter, and as a result, a flow velocity which is sufficient to resuspend the cells deposited in the tube and which does not cause cell deposition in the tube. It is possible to collect a small amount of culture solution.
Therefore, the number of times the culture solution is sampled can be significantly increased as compared with the conventional case, and continuous sampling is possible.
As a result, indexes such as glucose concentration, lactic acid concentration, cell concentration, and product concentration can be obtained more finely, and the culture can be controlled more accurately.

[Brief description of the drawings]

FIG. 1 is a perspective view showing an embodiment of a culture solution collecting device, and FIG.
The figure is a cross-sectional view showing a state in which the culture solution sampling device shown in FIG. 1 is mounted in a culture tank, and FIGS. 3 (a) and (b) and FIGS. 4 (a) and (b) are culture solution sampling devices. FIG. 5 shows another embodiment, FIGS. 5 (a), (b), (c) and (d) show how to use the culture solution sampling device, and FIG. 6 shows a conventional sample sampling device. It is a figure. FIG. 7 is a diagram showing the flow of the culture system according to the present invention. 1 ... Sampling pipe, 2a, 2b, 2c ... Steam distribution pipe, 3 ...
… Partition walls, 6,8,9 …… Flexible tubes, 10 …… Incubator.

Front page continuation (72) Inventor Masahiko Ishida 4026 Kuji Town, Hitachi City, Hitachi, Ibaraki Prefecture Hitachi Research Laboratory, Ltd. (72) Harumi Matsuzaki 4026 Kuji Town, Hitachi City, Ibaraki Hitachi Laboratory, Hitachi Research Center

Claims (5)

(57) [Claims] 1. An end of a culture solution collection tube having a smaller inner diameter is projected from the tip of a steam distribution tube, and the other end of the culture solution collection tube is larger than the outer diameter of the collection tube and is used for steam sterilization. An apparatus for collecting a culture solution, characterized by being housed in a steam flow pipe having an inner diameter that gives a sufficient cross-sectional area for allowing a steam flow rate capable of raising the temperature to a required temperature. 2. The steam flow tube according to claim 1, wherein the steam distribution tube has a pipe member a having a mechanism for connecting to a culture tank, and a pipe member b having a large inner diameter in the vicinity of the partition wall joined by a partition wall shared with the pipe member. Culture medium, which is composed of a tube material c connected to a tube material b at a large part of the medium, and a culture solution collecting tube penetrates a partition between the tube materials a and b and has one end opened inside the tube material. Sampling device. 3. The culture solution collecting device according to claim 1 or 2 is attached to a culture tank, steam is circulated in the culture solution collecting device to sterilize the inside of the collecting device, and then a tube material having a large inner diameter is obtained. A method for collecting a culture solution, which comprises removing a connected tube material and then collecting a sample through a sample collecting pipe housed in the tube material having a large inner diameter. 4. The method for collecting a culture solution according to claim 3, wherein the sample collecting pipe is connected to a pipe connected to a measuring device. 5. A culture tank equipped with a means for supplying gas to culture cells, a means for supplying a culture solution, and a means for discharging an aged culture solution, wherein one end of a culture solution collection tube having a small inner diameter is projected from the tip of the steam distribution tube. , Having an inner diameter that is larger than the outer diameter of the collection tube at the other end of the culture solution collection tube and that provides a sufficient cross-sectional area for passing a steam flow rate that can raise the temperature to the temperature required for steam sterilization A culture solution collection device housed in a steam distribution tube, a means for enlarging an image of cells in the culture solution collected by the culture solution collection device, and an image recognition device for recognizing live cells and dead cells from the image A continuous automatic diagnostic device for the number and activity of cells comprising: a lysed component automatic analyzer for analyzing lysed components such as glucose, lactic acid and ammonia in the culture solution collected by the culture solution collection device; Culture system, characterized in that it comprises a means for automatically operating the culture vessel diagnostic apparatus and the information from the dissolved components automatic analyzer based on.