JP7426065B2 - Cell fiber manufacturing system, cell fiber manufacturing method and program - Google Patents

Description

The present invention relates to a cell fiber manufacturing system, a cell fiber manufacturing method, and a program.

Methods of culturing cells on two-dimensional surfaces are well known. Instead, the following patents disclose systems for culturing and manufacturing cells in hollow hydrogel fibers made of alginate polymers. In this system, a cell solution containing cells is suspended in the hollow spaces of alginate hydrogel fibers, and then the hollow fibers containing cells are suspended in a cell culture medium. This allows cells to be cultured within the hollow fiber. Patent Document 1 describes that a wide variety of cells can be cultured on a large scale.

JP2018-534936 International Publication No. 2011/046105

The inventor of the present application has discovered that when cells are cultured within a fiber as described in Patent Document 1, cells leak from the fiber before or during cell culture depending on the production conditions of the cells and fiber. I found that there are some issues. Depending on the use of the produced cells, it is preferable to reduce the amount of cells leaking out of the fiber and to maintain the state in which the cells are confined within the fiber as much as possible.

Therefore, a cellular fiber manufacturing system and a cellular fiber manufacturing method that can suppress cell leakage from fibers are desired.

According to one aspect, the cell fiber manufacturing system includes a first channel through which a first fluid containing cells flows, and a second fluid for hydrogel preparation that joins around the first fluid in the first channel. and an ejection port that ejects at least the first fluid and the second fluid together. In the cell fiber manufacturing system, in the cell fiber manufacturing start stage, the first fluid is fed after the second fluid reaches at least a confluence point of the first flow path and the second flow path. It is configured like this.

According to another aspect, the method for producing cell fibers includes a first channel through which a first fluid containing cells flows and a second fluid for preparing a hydrogel, around the first fluid. The present invention relates to a cell fiber manufacturing method using a nozzle having a second flow path configured to flow along the flow, and an ejection port for ejecting at least the first fluid and the second fluid together. This cell fiber manufacturing method includes a step of feeding the second fluid at the start stage of cell fiber manufacturing, and after the second fluid reaches at least a confluence point of the first flow path and the second flow path. , and the step of feeding the first fluid.

According to another aspect, the program is a program that causes a computer to execute the above cell fiber manufacturing method.

According to the above aspect, leakage of cells from the fiber can be suppressed.

BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram which shows the whole structure of the cell fiber manufacturing system in 1st Embodiment. It is a figure which shows the flow chart of the cell fiber manufacturing method in 1st Embodiment. It is a graph showing temporal changes in the amounts of the first fluid, the second fluid, the third fluid, and the cleaning liquid in the first embodiment. FIG. 3 is a diagram showing a microscopic image of cell fibers in one embodiment. It is a figure which shows the flowchart of the cell fiber manufacturing method in 2nd Embodiment. It is a graph showing temporal changes in the amounts of the first fluid, the second fluid, and the cleaning liquid in the second embodiment. It is a schematic diagram showing the whole structure of a cell fiber manufacturing system in a 3rd embodiment. It is a figure which shows the flowchart of the cell fiber manufacturing method in 3rd Embodiment. It is a graph showing temporal changes in the amounts of the first fluid, the second fluid, and the cleaning liquid in the third embodiment.

Embodiments will be described below with reference to the drawings. In the following drawings, the same or similar parts are designated by the same or similar symbols.

[First embodiment]
FIG. 1 is a schematic diagram showing the overall configuration of a cell fiber manufacturing system in a first embodiment. The cell fiber manufacturing system 100 may include a first storage section 102, a second storage section 104, a third storage section 106, a nozzle 200, and a reservoir 300.

The first storage section 102 stores a first fluid containing cells. The first fluid may be, for example, a gel or liquid (suspension) containing cells. The types of these gels or liquids are not particularly limited. These gels or liquids may be, for example, chitosan gels, collagen gels, gelatin, peptide gels, fibrin gels, laminin gels, nanocellulose, pullulan, dextran, media, or alginic acid, or mixtures thereof.

The type of cells contained in the first fluid is not particularly limited. Such cells include, for example, ES cells and iPS cells that have pluripotency, various stem cells that have pluripotency (hematopoietic stem cells, neural stem cells, mesenchymal stem cells, etc.), and stem cells that have monopotency (such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells). Hepatic stem cells, reproductive stem cells, etc.). Instead, the cells contained in the first fluid may include various differentiated cells, such as muscle cells such as skeletal muscle cells and cardiomyocytes, nerve cells such as cerebral cortical cells, fibroblasts, epithelial cells, hepatocytes, It may also be pancreatic β cells, skin cells, etc. Furthermore, it should be noted that the "cells" contained in the first fluid are not limited to a single cell, but also include microorganisms such as bacteria in addition to cellular tissue consisting of a plurality of cells.

The first fluid contains various growth factors suitable for cell culture, cell maintenance and proliferation, and cell function expression, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor ( TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor (NGF), and the like.

The second storage section 104 stores a second fluid for hydrogel preparation (a solution that becomes the source of the hydrogel). Preferably, the second fluid is alginate or agarose. More specifically, the second fluid may be, for example, sodium alginate, potassium alginate, ammonium alginate, and combinations thereof. Moreover, alginic acid may be a natural extract or may be chemically modified. Examples of chemically modified alginic acid include methacrylate-modified alginic acid. Further, the second fluid may be a mixed system of the above-described alginate and agar, agarose, polyethylene glycol (PEG), polylactic acid (PLA), nanocellulose, or the like.

The third storage section 106 stores a third fluid that gels the second fluid when it comes into contact with the second fluid. When the aforementioned second fluid is the aforementioned alginate polymer material, the third fluid may be a solution containing polyvalent cations. Such solutions include, for example, solutions containing calcium or barium ions, such as calcium chloride or barium chloride.

The nozzle 200 may have a first inlet 210, a second inlet 220, a third inlet 230, and an injection port 240. The first inlet 210 is an inlet for a first fluid and is in fluid communication with the first reservoir 102 . The second inlet 220 is an inlet for a second fluid and is in fluid communication with the second reservoir 104 . The third inlet 230 is an inlet for a third fluid and is in fluid communication with the third reservoir 106 .

In this specification, the flow path from the first inlet 210 or the first storage section 102 to the injection port 240 is referred to as a "first flow path." That is, the first flow path 212 may be defined by a flow path through which the first fluid containing cells passes.

In this specification, the flow path from the second inlet 220 or the second storage section 104 to the confluence with the first flow path 212 is referred to as a "second flow path." That is, the second fluid passes through the second flow path 222 , joins the first flow path 212 , and then reaches the injection port 240 . Here, the confluence point of the first flow path 212 and the second flow path 222 is preferably located within the nozzle 200.

The second channel 222 is configured to allow a second fluid for hydrogel preparation to merge around the first fluid in the first channel 212 . Thereby, the second fluid flows around the flow of the first fluid along the direction of flow of the first fluid. That is, the second fluid surrounds the first fluid in a cross section perpendicular to the flow of the first fluid. More specifically, the first fluid and the second fluid preferably flow to form a laminar flow.

In this specification, the flow path from the third inlet 230 or the third storage section 106 to the confluence with the first flow path 212 is referred to as a "third flow path." That is, the third fluid passes through the third flow path 232 , merges with the first flow path 212 , and then reaches the injection port 240 . The third flow path 232 causes the third fluid to join the first flow path 212 on the downstream side of the confluence point of the first flow path 212 and the second flow path 222 . Here, the confluence point of the first flow path 212 and the third flow path 232 is preferably located within the nozzle 200.

The third fluid flows along the flow of the second fluid around the second fluid surrounding the first fluid on the downstream side of the confluence of the first flow path 212 and the third flow path 232. Preferably, the third fluid forms a laminar flow with the first fluid and the second fluid. The second fluid gels by contacting the third fluid. Therefore, the second fluid ejected from the injection port 240 is partially or completely gelled.

The diameters of the first flow path 212, the second flow path 222, and the third flow path 232 can be appropriately designed depending on the size of each portion of the cell fiber to be manufactured.

It is preferable that the first flow path 212 extends generally along the direction of gravity, at least on the downstream side of the confluence of the first flow path 212 and the second flow path 222. This makes it difficult for gravity to be applied in a direction perpendicular to the direction of flow of the first fluid, so that the second fluid flowing around the first fluid can move around the first fluid in a cross section perpendicular to the direction of flow of the first fluid. It becomes easier to cover with an even thickness.

It is preferable that the nozzle 200 is configured to be replaceable with respect to the cell fiber manufacturing apparatus 100. As a result, if the flow path in the nozzle 200 becomes clogged, the clogging can be eliminated by replacing the nozzle 200. In particular, the nozzle 200 portion includes a narrow flow path and is therefore more likely to become clogged than other portions of the device. Therefore, by configuring the nozzle 200 to be replaceable, the cell fiber manufacturing apparatus 100 can be used for a long period of time.

The cell fiber manufacturing system 100 includes a first pump 112 that pumps a first fluid toward an injection port 240, a second pump 114 that pumps a second fluid toward an injection port 240, and a third pump that pumps a third fluid. and a third pump 116 for delivering liquid toward the outlet 240. The first pump 112, the second pump 114, and the third pump 116 may be configured to be able to adjust the flow rates and flow rates of the first fluid, the second fluid, and the third fluid, respectively.

The flow rate settings for the first pump 112, the second pump 114, and the third pump 116 may be configured to be adjustable by the user, or may be automatically adjusted by a preset program. In this case, the cell fiber manufacturing system 100 may include a control unit that controls the various pumps 112, 114, 116, and 132.

The cell fiber manufacturing system 100 includes a first sensor 122 that detects the feeding state of the first fluid, a second sensor 124 that detects the feeding state of the second fluid, and a second sensor 124 that detects the feeding state of the third fluid. 3 sensors 126. The first sensor 122, the second sensor 124, and the third sensor 126 detect whether the first fluid, the second fluid, and the third fluid are flowing without clogging, respectively.

The cell fiber manufacturing system 100 may include a notification unit that notifies users of various information. The notification section may be, for example, a display, a light emitting section, or a buzzer. For example, when an abnormality such as clogging is discovered in the flow path by the first sensor 122, the second sensor 124, and/or the third sensor 126, the notification unit notifies the user. Such notification may be a warning displayed on a display, light emission or sound that indicates an abnormality, or the like.

The cell fiber manufacturing system 100 may include a reservoir 300 that receives the material injected from the injection port 240, and a stage 400 on which the reservoir 300 is mounted. During production of cellular fibers, reservoir 300 preferably contains a solution suitable for receiving cellular fibers. Such a solution may be, for example, saline.

Preferably, the cell fiber manufacturing system 100 includes an adjustment mechanism 410 that changes the distance between the reservoir 300 and the injection port 240. In this embodiment, the stage 400 is configured to be movable up and down. Alternatively, the nozzle 200 may be configured to be movable up and down.

The adjustment mechanism 410 allows the positional relationship between the injection port 240 and the liquid level in the reservoir 300 to be freely changed during the production of cell fibers. Here, during the production of cell fibers, the injection port 240 is preferably located below the liquid level in the reservoir 300, that is, in the liquid in the reservoir 300. Thereby, the cell fibers injected from the injection port 240 are directly received in the liquid, so that damage to the cell fibers can be suppressed.

The cell fiber manufacturing system 100 may include a detection unit capable of estimating or detecting that the injection port 240 is located within the liquid in the reservoir 300. Thereby, the adjustment mechanism 410 can automatically vary the distance between the reservoir and the injection port so that the injection port 240 is located within the liquid in the reservoir 300. Such a detection unit may be, for example, an imaging device (for example, a camera) provided in the cell fiber manufacturing system. The imaging device can detect contact between the liquid level in the reservoir and the nozzle outlet 240.

The cell fiber manufacturing system 100 may include a cleaning mechanism that cleans at least a portion of the first flow path 212, the second flow path 222, and the third flow path 232. In this embodiment, the cell fiber manufacturing system 100 may include a cleaning container 130 that stores a cleaning solution, and a cleaning pump 132 that can deliver the cleaning solution from the cleaning container 130. Preferably, the washing liquid is physiological saline.

The cleaning container 130 is preferably in fluid communication with at least one of the first flow path 212 , the second flow path 222 , and the third flow path 232 via a cleaning flow path 134 . That is, the cleaning channel 134 can allow the cleaning liquid to flow into at least one of the first channel 212, the second channel 222, and the third channel 232. Thereby, at least a portion of the first flow path and the second flow path can be cleaned via the cleaning flow path.

The cleaning pump 132 may be configured to send cleaning liquid from the cleaning channel 134 to the injection port 240. Thereby, the cleaning liquid can flow from the cleaning channel 134 to the injection port 240 via the first channel 212, the second channel 222, and/or the third channel 232. Therefore, clogging in the flow paths 212, 222, 232 can be suppressed. Further, there is an advantage that the flow channels 212, 222, and 232 can be cleaned without disassembling the cell fiber manufacturing system 100.

It is preferable that the cleaning channel 134 communicates with a position where the cleaning liquid can flow to a portion of the first channel 212, the second channel 222, and the third channel 232 that is likely to become clogged. In the embodiment shown in FIG. 1 , the cleaning channel 134 is configured to allow the cleaning liquid to flow into the third channel 232 . That is, the cleaning liquid reaches the injection port 240 from the cleaning container 130 via the cleaning channel 134 and the third channel 232.

In this case, the cleaning liquid can effectively clean at least a portion of the third flow path 232, particularly the vicinity of the confluence of the first flow path 212 and the third flow path 232, through the cleaning flow path 134. . The third flow path 232 is a flow path for a third fluid that gels the second fluid. If the second fluid unintentionally flows into the third flow path 232, the second fluid will gel within the third flow path 232. Therefore, clogging is likely to occur particularly near the confluence of the first flow path 212 and the third flow path 232. In this aspect, since the cleaning channel 134 communicates with the third channel 232, the cleaning liquid can suppress or eliminate clogging, especially near the confluence of the first channel 212 and the third channel 232. can.

In the embodiment shown in FIG. 1 , the cleaning channel 134 communicating with the cleaning container 130 communicates with the third channel 232 . Alternatively, the cleaning channel 134 communicating with the cleaning container 130 may communicate with the first channel 212 and/or the second channel 222. The cleaning channel 134 may be configured to allow the cleaning liquid to flow to a place where the channel is likely to become clogged. Further, the cleaning channel 134 that communicates with the cleaning container 130 may communicate with not only one of the first channel 212, the second channel 222, and the third channel 232, but also multiple locations. .

Preferably, a valve 150 is provided in the wash channel 134. Valve 150 may be, for example, an on-off valve or a one-way valve. As a result, the cleaning channel 134 is closed by the valve 150 at least while the cleaning liquid is not being fed. Therefore, it is possible to prevent the first fluid, the second fluid, and/or the third fluid from flowing back into the cleaning channel 134. In particular, in the embodiment shown in FIG. 1, it is possible to prevent the third fluid from flowing back into the cleaning channel 134 while the cleaning liquid is not being fed.

As shown in FIG. 1, a valve 170 may be provided in the third flow path 232. Valve 170 may be, for example, an on-off valve or a one-way valve. This prevents the third fluid from flowing out into the flow path 232 and the flow path 212 even after the third pump 116 is stopped, and the third flow path 232 is prevented from flowing out into the flow path 232 and the third flow path 232. Clogging due to gelation of the second fluid can be suppressed near the point. Valve 170 may be used in place of or in conjunction with the cleaning mechanisms described above. However, the valve 170 is not an essential component, and may be omitted if unnecessary.

[Cell fiber manufacturing method]
FIG. 2 is a diagram showing a flowchart of the cell fiber manufacturing method in the first embodiment. FIG. 3 is a graph showing changes over time in the amounts of the first fluid, the second fluid, the third fluid, and the cleaning liquid sent during the period from the start of cell fiber production to the end of production. Here, it should be noted that the vertical axis in FIG. 3 indicates the relative value of the amount of liquid sent. The cell fiber manufacturing method in the first embodiment can be realized using the cell fiber manufacturing system 100 described above.

First, a reservoir 300 containing a liquid such as physiological saline is placed on the stage 400 of the cell fiber manufacturing system 100, and the distance between the injection port 240 of the nozzle 200 and the reservoir 300 is adjusted (step S11). . Here, the height of the stage 400 is adjusted so that the injection port 240 of the nozzle 200 is immersed in the liquid in the reservoir 300.

Here, the cell fiber manufacturing system 100 automatically estimates or detects that the injection port 240 is located in the liquid in the reservoir 300, and automatically adjusts the distance between the reservoir 300 and the injection port 240 using the adjustment mechanism 410. It is preferable to vary it. However, the distance between the reservoir 300 and the injection port 240 may be manually set by the user.

Next, at the start stage of cell fiber production, a second fluid for hydrogel preparation and a washing liquid are sent toward the injection port 240 (step S12). The second fluid can be fed by the second pump 114. The cleaning liquid can be fed by the cleaning pump 132. At this time, as will be described later, it is preferable that the first fluid and the cleaning liquid form a laminar flow.

The cleaning liquid may be fed before the second fluid for hydrogel preparation, or may be fed at the same time as the second fluid for hydrogel preparation. If the second fluid is sent and the cleaning liquid is sent at the same time, the period of the cell fiber manufacturing start stage (preparation stage) can be reduced.

Next, after the second fluid reaches at least the confluence point of the first flow path and the second flow path, preferably after reaching the injection port 240, the feeding of the cleaning liquid is stopped, and the third fluid is Liquid feeding is started (step S13). The third fluid can be sent by the third pump 116. Note that the timing of stopping the feeding of the cleaning liquid and the timing of starting the feeding of the third fluid does not matter which comes first.

However, it is preferable that the feeding of the cleaning liquid is stopped at the same time as the feeding of the third fluid is started. As a result, the period required for the process of stopping the supply of the cleaning liquid and starting the supply of the third fluid is reduced.

More preferably, the amount of the third fluid sent is gradually increased while the amount of the cleaning liquid sent is gradually decreased. Thereby, it is possible to suppress the second fluid from entering the third flow path 232 during the process of stopping the cleaning liquid feeding and starting the third fluid feeding. Furthermore, in this case, it is more preferable to form a laminar flow with the second fluid and the cleaning liquid, and then gradually decrease the amount of the cleaning liquid sent and gradually increase the amount of the third fluid sent. Thereby, the flow of the cleaning liquid can be replaced with the flow of the third fluid while maintaining the state in which the laminar flow is formed. Therefore, in the process of starting to flow the third fluid, the possibility that the second fluid and the third fluid mix due to turbulence is reduced, and the possibility that the flow path in the nozzle 200 is clogged by the gelled second fluid is further reduced. I can do it.

In step S13, the third fluid is configured to be delivered after the second fluid reaches at least the confluence point of the third flow path 232 and the first flow path 212, particularly after reaching the injection port 240. It is preferable.

Next, at the start stage of cell fiber production, both the second fluid and the third fluid preferably reach the injection port 240 after reaching at least the confluence of the third flow path 232 and the first flow path 212. Afterwards, feeding of the first fluid is started (step S14). The first fluid can be fed by the first pump 112.

Thereby, after the second fluid and the third fluid join together, the first fluid is sent. That is, after the second fluid is partially or completely gelled by the third fluid, the first fluid containing the cells begins to merge with the second fluid and the third fluid. That is, the first fluid containing cells is delivered after the tip portion of the cell fiber is partially or completely gelled. Thereby, leakage of cells from the tip of the fiber can be further suppressed. In particular, even when cells are cultured within the cell fibers, leakage of the cells from the tips of the cell fibers can be suppressed.

Next, the feeding of the first fluid, the second fluid, and the third fluid is maintained for a predetermined period (step S15). During this period, a substance generated by the merging of the first fluid, the second fluid, and the third fluid is injected from the injection port 240. Specifically, the object to be injected is a cell containing a generally tubular hydrogel generated by the confluence of a second fluid and a third fluid, and a first fluid containing cells filled in the hydrogel. It is fiber. The length of the cell fibers can be adjusted depending on the length of the liquid feeding period in step S15.

In the step of stopping the production of cell fibers, first, the feeding of the first fluid is stopped (step S16). Even after stopping the feeding of the first fluid, it is preferable to continue feeding the second fluid and the third fluid for a predetermined period of time. This causes the rear end portion of the cell fiber to be sufficiently closed with the (gelled) second fluid. Thereby, cells within the cell fiber can be suppressed from leaking out from the rear end of the fiber.

Next, in the step of stopping the production of cell fibers, the feeding of the third fluid is stopped, and the feeding of the cleaning liquid is started (step S17). It is preferable that the feeding of the cleaning liquid is stopped at the same time as the feeding of the third fluid is started. As a result, the fluid flowing inside the nozzle 200 is gradually replaced by the cleaning liquid from the third fluid. As a result, the third fluid within the nozzle 200 is forced out of the injection port 240 by the cleaning liquid. This prevents the second fluid from coming into contact with the third fluid, suppressing gelation of the second fluid, and suppressing clogging during the stop stage.

Next, in the step of stopping the production of cell fibers, the feeding of the second fluid and the washing liquid are stopped (step S18). The feeding of the cleaning liquid may be stopped before the feeding of the second fluid is stopped, or may be after the feeding of the second fluid is stopped. Further, the feeding of the cleaning liquid may be stopped at the same time as the feeding of the second fluid is stopped.

Preferably, the feeding of the cleaning liquid is stopped at the same time as, or after, the feeding of the second fluid is stopped. Thereby, it is possible to suppress the second fluid from flowing back into the third flow path 232.

In the method described above, in the step of stopping production of cell fibers, the feeding of the second fluid is stopped after the feeding of the third fluid is stopped. In this case, there is a period during which the second fluid flows even after the third fluid that causes the second fluid to gel is stopped. Therefore, there is a period in which the second fluid forces the residue of the third fluid out of the injection port 240. Therefore, it is possible to prevent the gelled second fluid from remaining in the nozzle 200 and causing clogging after the production of cell fibers is stopped.

After the aforementioned steps S11 to S18, the cell fibers generated in the reservoir 300 may be replaced into the culture medium. In this case, since the cells are confined within the cell fibers, the cells can be easily transferred into the culture medium. Furthermore, since cells are hardly mixed into the waste liquid in the reservoir 300, the waste liquid can be easily disposed of.

The cells within the cell fibers may be cultured for a predetermined period of time while being transferred into a culture medium. Since both ends of the fiber in the extending direction are closed with hydrogel, even when the number of cells increases, the cells remain confined within the cell fiber.

At the above-mentioned cell fiber production start stage or stop stage, whether the cleaning liquid, the second fluid and/or the third fluid has reached any position in the flow path or the injection port 240 can be determined by the human eye or by the human eye or the cell fiber manufacturing stage. This can be determined manually or automatically by the manufacturing system. For example, by mounting a camera on the cell fiber manufacturing system and using the camera to detect whether the cleaning liquid, the second fluid, and/or the third fluid has been injected from the injection port 240, the cell fiber manufacturing system automatically determines be able to. Instead, the cell fiber manufacturing system uses the first sensor 122, the second sensor 124, and the third sensor 126 described above, so that the cleaning liquid, the second fluid, and/or the third fluid can reach any part of the flow path. It may be determined whether the position or exit port 240 has been reached. In this case, the cell fiber manufacturing system is capable of producing various fluids based on the distance between the sensors 122, 124, 126 and any position in the flow path or the injection port 240, and the flow rate of the various fluids. What is necessary is to estimate the time it takes from the sensors 122, 124, 126 to reach any position in the flow path or the injection port 240. Thereby, the cell fiber manufacturing system uses the first sensor 122, the second sensor 124, and the third sensor 126 to control the cleaning liquid, the second fluid, and/or the third fluid at any position or injection position in the flow path. Whether exit 240 has been reached can be automatically determined.

FIG. 4 is a diagram illustrating a microscopic image of cell fibers in one embodiment. FIG. 4 is a micrograph of cell fibers produced by the apparatus and method described above. FIG. 4 shows the state after cells have been cultured within the cell fiber for a long period of time.

This cell fiber includes a plurality of cells 800 lined up along the fiber, and a hydrogel 810 that covers the cells in a cross section intersecting the extending direction of the fiber. The hydrogel 810 is closed at both ends 820 of the fiber to confine the cells. This hydrogel 810 is composed of the second fluid gelled by the third fluid described above. In the example shown in FIG. 4, sodium alginate was used as the second fluid and calcium chloride was used as the third fluid. Furthermore, in the example shown in FIG. 4, the cells in the first fluid are iPS cell-derived cardiomyocytes.

The outer diameter of the cell fibers is not particularly limited, but may range, for example, from about 1 μm to 5 mm. The length of the cell fibers is not particularly limited. The length of the cell fiber may be, for example, about 20 mm to 100 cm. The inner diameter of the hydrogel (outer shell) constituting the cell fiber is not particularly limited, but may range, for example, from about 100 nm to 1000 μm. Further, at both ends of the cell fiber in the extending direction, the length of the region of the gel portion that does not contain cells is preferably, for example, 10 times or more the outer diameter of the cell fiber. For example, at both ends of the cell fiber in the extending direction, the length of the region of the gel portion that does not contain cells may be greater than 1 mm, preferably 5 mm, and more preferably greater than 20 mm. Thereby, cells within the fiber can be suppressed from leaking from the end of the fiber.

[Second embodiment]
FIG. 5 is a diagram showing a flowchart of the cell fiber manufacturing method in the second embodiment. FIG. 6 is a graph showing changes over time in the amounts of the first fluid, the second fluid, the third fluid, and the cleaning liquid sent during the period from the start of cell fiber production to the end of production. It should be noted here that the vertical axis in FIG. 6 indicates the relative value of the amount of liquid sent. The cell fiber manufacturing method in the second embodiment can be realized, for example, using the cell fiber manufacturing system 100 described in the first embodiment (see also FIG. 1).

First, a reservoir 300 containing a liquid such as physiological saline is placed on the stage 400 of the cell fiber manufacturing system 100, and the distance between the injection port 240 of the nozzle 200 and the reservoir 300 is adjusted (step S31). . Step S31 is similar to step S11 in the first embodiment.

Next, at the stage of starting production of cell fibers, a cleaning liquid is sent toward the injection port 240 as necessary (step S32). The cleaning liquid can be fed by the cleaning pump 132.

By flowing the cleaning liquid before feeding the first fluid, the second fluid, or the third fluid, it is possible to push out what remains in the flow path. In particular, if the third fluid flows in while the second fluid remains in the channel within the nozzle 200, the second fluid may gel and cause clogging. By feeding the cleaning liquid first, the possibility of such clogging can be reduced.

Next, the feeding of the cleaning liquid is stopped, and a third fluid for gelling the second fluid is fed toward the injection port 240 (step S33). The third fluid can be sent by the third pump 116.

Next, the second fluid for hydrogel preparation is sent toward the injection port 240 (step S34). The second fluid can be fed by the second pump 114. The feeding of the second fluid is started after the third fluid reaches at least the confluence of the third flow path 232 and the second flow path 222, preferably after reaching the injection port 240. More preferably, feeding of the second fluid is started after the flow of the third fluid is stabilized. In step S34, it is preferable that a laminar flow is formed between the second fluid and the third fluid.

Next, at the start stage of cell fiber production, both the second fluid and the third fluid preferably reach the injection port 240 after reaching at least the confluence of the third flow path 232 and the first flow path 212. Afterwards, feeding of the first fluid is started (step S35). The first fluid can be fed by the first pump 112.

Thereby, after the second fluid and the third fluid join together, the first fluid is sent. That is, after the second fluid is partially or completely gelled by the third fluid, the first fluid containing the cells begins to merge with the second fluid and the third fluid.

Next, the feeding of the first fluid, the second fluid, and the third fluid is maintained for a predetermined period (step S36). Step S36 is similar to step S15 in the first embodiment.

Next, in the step of stopping the production of cell fibers, first, the feeding of the first fluid is stopped (step S37). Even after stopping the feeding of the first fluid, it is preferable to continue feeding the second fluid and the third fluid for a predetermined period of time. This causes the rear end portion of the cell fiber to be sufficiently closed with the (gelled) second fluid. Thereby, cells within the cell fiber can be suppressed from leaking out from the rear end of the fiber.

Next, in the step of stopping the production of cell fibers, the feeding of the second fluid is stopped (step S38). Next, feeding of the third fluid is stopped (step S39). Alternatively, the feeding of the second fluid may be stopped after the feeding of the third fluid is stopped.

Next, the cleaning liquid is sent toward the injection port 240 (step S40). By feeding the cleaning liquid after stopping the feeding of the second fluid and the third fluid, it is possible to suppress residue from remaining in the flow path within the nozzle 200.

In the second embodiment, in the manufacturing start stage, the third fluid is started to be fed, and then the second fluid is started to be fed. Alternatively, in the manufacturing start stage, the second fluid and the third fluid may start to be fed at substantially the same timing.

[Third embodiment]
FIG. 7 is a schematic diagram showing the overall configuration of a cell fiber manufacturing system in the third embodiment. In FIG. 7, the same components as in the first embodiment are denoted by the same reference numerals. Furthermore, descriptions of configurations similar to those in the first embodiment may be omitted.

The cell fiber manufacturing system 100 may include a first storage section 102, a second storage section 104, a nozzle 200, and a reservoir 300. The first fluid stored in the first storage section 102 is the same as in the first embodiment. The second fluid stored in the second storage section 104 is the same as in the first embodiment.

In the third embodiment, the cell fiber manufacturing system 100 does not include the third storage section 106 or the third flow path 232. A third fluid that gels the second fluid is placed in the reservoir 300 during the production of the cell fibers. Note that the material of the third fluid may be the same as in the first embodiment.

In the third embodiment, the ejection port 240 ejects the first fluid and the second fluid together into a third fluid within the reservoir 300. Thereby, the second fluid comes into contact with the third fluid within the reservoir 300 and is gelled.

The cell fiber manufacturing system 100 may include a cleaning mechanism that cleans at least a portion of the first flow path 212 and the second flow path 222. In the third embodiment, the cleaning channel 134 is configured to communicate with the second channel 222 and allow the cleaning liquid to flow into the second channel 222 . Alternatively, the cleaning channel 134 may be configured to communicate with the first channel 212 and allow the cleaning liquid to flow into the first channel 212.

[Cell fiber manufacturing method]
FIG. 8 is a diagram showing a flowchart of the cell fiber manufacturing method in the third embodiment. FIG. 9 is a graph showing changes over time in the amounts of the first fluid, the second fluid, and the cleaning liquid sent during the period from the start of cell fiber production to the end of production. Here, it should be noted that the vertical axis in FIG. 9 indicates the relative value of the amount of liquid sent. The cell fiber manufacturing method in the third embodiment can be realized using the cell fiber manufacturing system 100 shown in FIG. 7.

First, a reservoir 300 containing a liquid such as physiological saline is placed on the stage 400 of the cell fiber manufacturing system 100, and the distance between the injection port 240 of the nozzle 200 and the reservoir 300 is adjusted (step S21). . Here, the height of the stage 400 is adjusted so that the injection port 240 of the nozzle 200 is immersed in the third fluid in the reservoir 300.

Here, the cell fiber manufacturing system 100 automatically estimates or detects that the injection port 240 is located in the third fluid in the reservoir 300, and the adjustment mechanism 410 automatically adjusts the distance between the reservoir 300 and the injection port 240. Preferably, the distance is varied. However, the distance between the reservoir 300 and the injection port 240 may be manually set by the user.

Next, at the stage of starting production of cell fibers, a cleaning liquid is sent toward the injection port 240 (step S22). Next, the feeding of the cleaning liquid is stopped, and the second fluid for hydrogel preparation is fed toward the injection port 240 (step S23).

Next, after the second fluid reaches at least the confluence point of the first flow path and the second flow path, preferably after reaching the injection port 240, feeding of the first fluid is started (step S24). . At this time, it is preferable that the first fluid and the second fluid form a laminar flow. Since the first fluid containing cells is delivered after the second fluid, the tip portion of the cell fiber is sufficiently closed with the (gelled) second fluid. Thereby, it is possible to suppress leakage of cells from the tip of the fiber during production of the cell fiber.

Next, the feeding of the first fluid and the second fluid is maintained for a predetermined period (step S25). During this period, a confluence of the first fluid and the second fluid is injected from the injection port 240. The second fluid contacts the third fluid within the reservoir 300 and is gelled. This produces a cellular fiber that includes a generally tubular hydrogel and a first fluid containing cells filled within the hydrogel. The length of the cell fibers can be adjusted depending on the length of the liquid feeding period in step S25.

In the step of stopping the production of cell fibers, first, the feeding of the first fluid is stopped (step S26). Even after stopping the feeding of the first fluid, it is preferable to continue feeding the second fluid for a predetermined period of time. This causes the rear end portion of the cell fiber to be sufficiently closed with the (gelled) second fluid. Therefore, cells within the cell fiber can be prevented from leaking out from the rear end of the fiber.

Next, in the step of stopping the production of cell fibers, the feeding of the second fluid is stopped, and the feeding of the cleaning liquid is started (step S27). Thereby, the inside of the nozzle 200 can be cleaned with the cleaning liquid at the stage of stopping the production of cell fibers.

Next, in the step of stopping the production of cell fibers, the feeding of the cleaning solution is stopped (step S28). Similar to the first embodiment, after the aforementioned steps S21 to S28, the cell fibers generated in the reservoir 300 may be replaced in the culture medium.

As described above, the content of the present invention has been disclosed through the embodiments, but the statements and drawings that form part of this disclosure should not be understood as limiting the present invention. Various alternative embodiments, implementations, and operational techniques will be apparent to those skilled in the art from this disclosure. Therefore, the technical scope of the present invention is determined only by the matters specifying the invention in the claims that are reasonable from the above description.

For example, the cell fiber manufacturing method in the first embodiment, second embodiment, and third embodiment described above includes a step of feeding a washing liquid at the start stage or the stop stage. However, the step of sending the cleaning liquid is not an essential step and may be performed as necessary. Specifically, the step of sending the cleaning liquid may not be performed at one or both of the production start stage and stop stage. Even if cleaning is not performed in one or both of the manufacturing start stage and the stop stage, the order of flowing the first fluid, second fluid, and/or third fluid may be performed as described above. Even if there is no step of sending a cleaning solution between the production start and stop stages, the cell fiber production system is equipped with the above-mentioned cleaning channel 134, etc., so that cleaning can be performed at the appropriate timing, such as during maintenance. It is preferable that

Also, it should be noted that the various configurations described in each embodiment can be combined and/or replaced as much as possible. For example, in the cell fiber manufacturing method, the process at the manufacturing start stage described in the first embodiment and the process at the manufacturing stop stage described in the second embodiment may be combined. Furthermore, the process at the manufacturing start stage described in the second embodiment and the process at the manufacturing stop stage described in the third embodiment may be combined.

The flowcharts shown in FIGS. 2, 5, and 8 may be performed manually or semi-manually by a person. For example, the settings of the cleaning pump 132, the first pump 112, the second pump 114, and the third pump 116 may be switched manually by a person. Alternatively, the setting conditions of the cleaning pump 132, the first pump 112, the second pump 114, and the third pump 116 may be automatically switched by a computer, for example, a computer installed in the cell fiber manufacturing apparatus. In this case, the setting conditions of the various pumps, ie, the liquid feeding protocol, may be determined in advance by a program. In particular, it is preferable that the liquid delivery protocol at the cell fiber production start stage and/or the cell fiber production stop stage be executed according to a predetermined program.

It should be noted that the scope of the present invention also includes a program that causes a computer to execute the above-described cell fiber manufacturing method described with reference to the flowcharts shown in FIGS. 2, 5, and 8. Such computer programs can be stored on various types of non-transitory computer readable media and provided to the computer. Non-transitory computer-readable media include various types of tangible storage media. Examples of non-transitory computer-readable media include magnetic recording media (eg, flexible disks, magnetic tapes, hard disk drives), magneto-optical recording media (eg, magneto-optical disks), CD-ROMs (Read Only Memory), CD-Rs, CD-R/W, semiconductor memory (for example, mask ROM, PROM (programmable ROM), EPROM (erasable PROM), flash ROM, RAM (random access memory)). The program may also be provided to the computer on various types of transitory computer readable media. Examples of transitory computer-readable media include electrical signals, optical signals, and electromagnetic waves. The temporary computer-readable medium can provide the program to the computer via wired communication channels, such as electrical wires and fiber optics, or wireless communication channels.

It can be understood from the embodiments described above that at least the following inventions are also explained.

The cell fiber manufacturing system according to one embodiment includes a first flow path through which a first fluid containing cells flows, and a second flow path through which a second fluid for hydrogel preparation is merged around the first fluid in the first flow path. It has two flow paths and an ejection port that ejects at least the first fluid and the second fluid together. In the cell fiber manufacturing system and/or method, in the cell fiber manufacturing start stage, after the second fluid reaches at least a confluence point of the first flow path and the second flow path, the first fluid It is configured to send liquid. Preferably, the cell fiber manufacturing system and/or method is configured such that the first fluid is delivered after the second fluid reaches the injection port in the cell fiber manufacturing start stage.

Since the first fluid containing cells is delivered after the second fluid, the tip portion of the cell fiber is sufficiently closed with the (gelled) second fluid. Thereby, it is possible to suppress leakage of cells from the tip of the fiber during production of the cell fiber. Note that when cells leak from a cell fiber, it is not always easy to determine where the cells leaked from. It should be noted that the present inventors have discovered that cells leak out from the tip or rear end of cell fibers depending on the conditions, leading to the present invention.

A cell fiber manufacturing system and/or method according to another aspect is configured to stop feeding the second fluid after stopping the feeding of the first fluid in the step of stopping production of the cell fiber.

In this embodiment, since the second fluid is fed even after the feeding of the first fluid containing cells is stopped, the rear end portion of the cell fiber is sufficiently closed with the (gelled) second fluid. Become. Thereby, it is possible to suppress leakage of cells from the rear end of the fiber during production of the cell fiber.

According to another aspect, the cell fiber manufacturing system and/or method supplies a third fluid that gels the second fluid to a downstream side of a confluence point of the first flow path and the second flow path. and has a third flow path that merges with the first flow path.

In this embodiment, before the first fluid containing cells and the second fluid for hydrogel preparation are ejected from the injection port, the second fluid can be partially or completely gelled with the third fluid. Therefore, since the cell fibers can be ejected from the injection port in a more stable state, damage to the cell fibers can be suppressed. Additionally, when a third fluid that gels the second fluid is merged into the nozzle, there is an advantage that the thickness of the cell fibers can be adjusted by adjusting the flow rate of the third fluid. .

According to another aspect, in the cell fiber manufacturing system and/or method, in the cell fiber manufacturing start stage, both the second fluid and the third fluid are connected to at least the third flow path and the first flow path. The first fluid is configured to be delivered after reaching the confluence point of the first fluid. Preferably, in the cell fiber manufacturing system and/or method, the first fluid is delivered after both the second fluid and the third fluid reach the injection port in the cell fiber manufacturing start stage. It is configured like this.

In this aspect, the first fluid is fed after the second fluid and the third fluid join together. That is, the first fluid containing cells is delivered after the tip portion of the cell fiber is partially or completely gelled. Therefore, leakage of cells from the tip of the fiber can be further suppressed during production of the cell fiber.

According to another aspect, in the cell fiber manufacturing system and/or method, after the second fluid reaches the confluence of the third flow path and the first flow path in the cell fiber manufacturing start stage, , the third fluid is configured to be delivered. Preferably, the cell fiber manufacturing system and/or method is configured such that the third fluid is delivered after the second fluid reaches the injection port in the cell fiber manufacturing start stage.

In this aspect, since the third fluid is allowed to flow after the flow of the second fluid is stabilized, it becomes easier to form a laminar flow between the second fluid and the third fluid. This reduces the possibility that the second fluid and the third fluid will mix with each other due to turbulence, and it is possible to reduce the possibility that the flow path in the nozzle 200 will be clogged by the gelled second fluid.

According to another aspect, in the cell fiber manufacturing system and/or method, in the step of stopping the production of cell fibers, after stopping the feeding of the first fluid, the feeding of the second fluid and the third fluid is stopped. Configured to stop.

In this aspect, even when the feeding of the first fluid containing cells is stopped, the second fluid and the third fluid are combined and ejected. That is, the tip portion of the cell fiber is partially or completely gelled, and the rear end portion is closed by the hydrogel. Therefore, leakage of cells from the rear end of the fiber can be further suppressed.

According to another aspect, the cell fiber manufacturing system and/or method is configured to stop feeding the second fluid after stopping the feeding of the third fluid in the step of stopping the production of cell fibers. ing.

In this aspect, there is a period during which the second fluid flows even after the third fluid that causes the second fluid to gel is stopped. The second fluid will therefore force the remainder of the third fluid out of the injection port. Therefore, it is possible to prevent the gelled fluid from remaining in the channel and causing clogging after the production of cell fibers is stopped.

According to another aspect, the cell fiber manufacturing system and/or method includes a washing channel that allows a washing liquid to flow into at least one of the first channel and the second channel.

In this aspect, at least a portion of the first flow path and the second flow path can be cleaned via the cleaning flow path. Therefore, clogging of the flow path can be suppressed. Another advantage is that the flow path can be cleaned without disassembling the cell fiber manufacturing system.

According to another aspect, the cell fiber manufacturing system and/or method includes a washing channel that allows a washing liquid to flow into the third channel.

In this aspect, at least a portion of the third channel can be cleaned via the cleaning channel. The third flow path is a flow path for a third fluid that gels the second fluid. If the second fluid unintentionally flows into the third flow path, the second fluid will gel in the third flow path. This may cause clogging in the third flow path. Since the cleaning channel communicates with the third channel, such clogging in the third channel can be eliminated by the cleaning liquid.

According to another aspect, the cell fiber manufacturing system and/or method includes a liquid feeding pump that feeds the cleaning liquid from the cleaning channel to the injection port.

In this aspect, the liquid feeding pump can flow the cleaning liquid from the cleaning channel to the injection port via the first channel, the second channel, and/or the third channel. In addition, since the third fluid causes the second fluid to gel, the flow path through which the third fluid passes is likely to become clogged. Therefore, it is more preferable that the cleaning liquid is configured to pass through the confluence of the third flow path and the first flow path and downstream of the confluence.

According to another aspect, the cell fiber manufacturing system and/or method is configured to feed the cleaning liquid at least before the start of feeding the first fluid in the cell fiber manufacturing start stage. .

In this embodiment, production of cell fibers can be started after cleaning the channel within the nozzle.

According to another aspect, the cell fiber manufacturing system and/or method is configured to feed the washing liquid at least after stopping the feeding of the first fluid in the step of stopping the production of cell fibers.

In this aspect, since the channel in the nozzle can be cleaned at the stage of stopping production of cell fibers, it is possible to suppress clogging in the channel after the production is stopped.

According to another aspect, the cell fiber manufacturing system and/or method is configured to feed the cleaning liquid at least at the start of or before the start of feeding the third fluid in the cell fiber manufacturing start stage. has been done.

In this embodiment, gelling of the second fluid can begin after cleaning the flow path within the nozzle. That is, the flow path within the nozzle can be cleaned before generating the hydrogel.

According to another aspect, the cell fiber manufacturing system and/or method is configured to feed the cleaning liquid at least when or after the feeding of the third fluid is stopped in the step of stopping the production of cell fibers. ing.

In this aspect, it is possible to suppress the second fluid from flowing back into the third channel by feeding the cleaning liquid during the production stop stage. Moreover, even if the second fluid mixes into the third flow path and becomes a gel within the third flow path, it can be pushed out from the injection port by feeding the cleaning liquid. Thereby, clogging in the nozzle 200 can be further suppressed.

According to another aspect, the cell fiber manufacturing system and/or method includes a valve provided in the washing channel. The valve may be, for example, an on-off valve or a one-way valve.

In this aspect, the cleaning channel is closed by the valve at least while the cleaning liquid is not being sent, so it is possible to prevent the first fluid, the second fluid, and/or the third fluid from flowing back into the cleaning channel. .

According to another aspect, the cell fiber manufacturing system and/or method includes a valve provided in the third flow path. The valve may be an on-off valve or a one-way valve.

In this aspect, it is possible to prevent the second fluid from flowing back into the third flow path. Thereby, it is possible to suppress clogging of the third flow path near the confluence point of the first flow path and the third flow path due to gelation of the second fluid.

According to another aspect, the cell fiber manufacturing system and/or method includes a reservoir that receives an object ejected from the injection port, and an adjustment mechanism that changes the distance between the reservoir and the injection port. .

In this aspect, the positional relationship between the injection port and the liquid level in the reservoir can be freely changed during production of the cell fiber.

According to another aspect, the cellular fiber manufacturing system and/or method includes a detection unit capable of estimating or detecting that the injection port is located within the liquid in the reservoir.

In this embodiment, it is possible to detect that the injection port is located within the liquid in the reservoir prior to production of the cell fiber. Thereby, the adjustment mechanism can automatically vary the distance between the reservoir and the ejection port such that the ejection port is located within the liquid in the reservoir.

According to another aspect, the cell fiber manufacturing system and/or method includes a nozzle including the first channel, the second channel, the third channel, and the injection port, and the nozzle is replaceable. It is composed of

In this aspect, if the flow path in the nozzle 200 becomes clogged, the clog can be easily eliminated by replacing the nozzle. Therefore, by configuring the nozzle to be replaceable, the cell fiber manufacturing system can be used for a long period of time.

According to another aspect, a nozzle for a cell fiber manufacturing system includes a first channel through which a first fluid containing cells flows, and a second fluid for preparing a hydrogel through the first fluid in the first channel. and a third fluid that gels the second fluid into the first flow path downstream of the confluence point of the first flow path and the second flow path. and an injection port that discharges at least the first fluid, the second fluid, and the third fluid together. This nozzle is configured to be replaceable with respect to the cell fiber manufacturing system.

According to another aspect, the cellular fiber includes a plurality of cells arranged along the fiber and a hydrogel surrounding the cells in a cross section intersecting the extending direction of the fiber. The hydrogel is closed at both ends of the fiber in order to confine the cells.

In this embodiment, the cells are closed by the hydrogel at both the front and back ends of the fiber. Therefore, even if cells are cultured within the fiber, leakage of the cells from the tip or rear end of the fiber can be suppressed during production of the cell fiber.

100 Cell manufacturing system 102 First storage section 104 Second storage section 106 Third storage section 130 Washing container 132 Washing pump 134 Washing channel 200 Nozzle 210 First inlet 212 First channel 220 Second inlet 222 Second channel 230 Third inlet 232 Third flow path 240 Injection port 300 Reservoir 400 Stage

Claims (12)

a first channel through which a first fluid containing cells flows;
a second flow path in which a second fluid for hydrogel preparation is merged around the first fluid in the first flow path;
an ejection port that ejects at least the first fluid and the second fluid together;
a cleaning channel that allows cleaning liquid to flow into at least one of the first channel and the second channel,
The washing channel is at least one of the first channel and the second channel, and communicates with the upstream side of the confluence of the first channel and the second channel. Fiber manufacturing system. a first channel through which a first fluid containing cells flows;
a second flow path in which a second fluid for hydrogel preparation is merged around the first fluid in the first flow path;
a third flow path that causes a third fluid that gels the second fluid to join the first flow path downstream of a confluence point of the first flow path and the second flow path;
an injection port that discharges the first fluid, the second fluid, and the third fluid together;
a cleaning channel that allows a cleaning liquid to flow into at least one of the first channel, the second channel, and the third channel,
The cleaning channel is at least one of the first channel, the second channel, and the third channel, and is located upstream of the confluence of the first channel and the third channel. A cell fiber manufacturing system connected to . The cleaning channel according to claim 1 or 2, wherein the cleaning channel is not provided in a portion of the first channel upstream of a confluence point of the first channel and the second channel . Cell fiber manufacturing system. 4. The cell fiber manufacturing system according to claim 2, wherein the cleaning liquid is fed at least at the start of or before the start of feeding the third fluid in the cell fiber manufacturing start stage. The cell fiber manufacturing system according to any one of claims 2 to 4, wherein the washing liquid is fed at least when or after feeding of the third fluid is stopped in the step of stopping the production of cell fibers. The cell fiber manufacturing system according to any one of claims 2 to 5, comprising a valve provided in the third flow path. a reservoir for receiving the material ejected from the injection port;
The cell fiber manufacturing system according to any one of claims 1 to 6, further comprising an adjustment mechanism that changes the distance between the reservoir and the injection port. The cell fiber manufacturing system according to claim 7, further comprising a detection unit capable of estimating or detecting that the injection port is located in the liquid in the reservoir. a nozzle including the first flow path, the second flow path, the third flow path, and the injection port;
The cell fiber manufacturing system according to any one of claims 2 to 6, wherein the nozzle is configured to be replaceable. A nozzle for a cell fiber manufacturing system according to any one of claims 1 to 9,
a first channel through which a first fluid containing cells flows;
a second flow path in which a second fluid for hydrogel preparation is merged around the first fluid in the first flow path;
an ejection port that ejects at least the first fluid and the second fluid together;
A nozzle, comprising: a cleaning channel that allows a cleaning liquid to flow into at least one of the first channel and the second channel. A cleaning method using the cell fiber manufacturing system according to claim 1, comprising:
The cleaning liquid is caused to flow into at least one of the first flow path and the second flow path from the cleaning flow path, and the cleaning liquid is flowed into at least a confluence point of the first flow path and the second flow path. The cleaning method includes flowing the water through the injection port . A cleaning method using the cell fiber manufacturing system according to claim 2,
The cleaning liquid is caused to flow into at least one of the first flow path, the second flow path, and the third flow path from the cleaning flow path, and the cleaning liquid is caused to flow into at least one of the first flow path and the third flow path. A cleaning method comprising flowing the flow to the injection port through a confluence point with a flow path..

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