JP6848145B2 - Fiber manufacturing system, fiber manufacturing method and program - Google Patents

Description

The present invention relates to fiber manufacturing systems, fiber manufacturing methods and programs.

A well-known method is to culture cells on a two-dimensional surface. Instead, Patent Documents 1 and 2 below disclose a system for culturing and producing cells in hollow hydrogel fibers made of alginate polymers. In this system, a cell solution containing cells is suspended in the hollow space of hydrogel fibers of alginate, and then the hollow fibers containing cells are suspended in a cell culture medium. This causes the cells to be cultured in the hollow fiber. Patent Document 1 describes that a wide variety of cells can be cultured on a large scale.

JP-A-2018-534936 International Publication No. 2011/046105

When the cells are cultured in the fiber as described in Patent Document 1, the inventor of the present application leaks the cells from the fiber before or during the culture of the cell, depending on the conditions for producing the cell and the fiber. I found the problem that there are times. Depending on the intended use of the produced cells, it is preferable to reduce the amount of cells leaking from the fiber and keep the cells confined in the fiber as much as possible.

Therefore, a fiber manufacturing system or a fiber manufacturing method capable of suppressing cell leakage from the fiber is desired.

According to one aspect, the fiber manufacturing system merges a first fluid through which the first fluid containing cells flows and a second fluid for hydrogel preparation around the first fluid in the first fluid. A third fluid having a second flow path and at least an injection port for discharging the first fluid and the second fluid together, and the second fluid gels the second fluid at the production start stage. The first fluid is configured to begin merging with the second fluid after merging with.

According to another aspect, the fiber manufacturing system puts a first fluid through which a first fluid containing cells flows and a second fluid for hydrogel preparation around the first fluid in the first fluid. This is a fiber manufacturing method using an apparatus having a second flow path to be merged and at least an injection port for discharging the first fluid and the second fluid together. In the present fiber manufacturing method, the first fluid is so as to start merging with the second fluid after the second fluid merges with the third fluid that gels the second fluid at the manufacturing start stage. The fluid and the second fluid are sent.

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

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

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 flowchart of the cell fiber manufacturing method in 1st Embodiment. It is a graph which shows the time change of the liquid feed amount of the 1st fluid, the 2nd fluid, the 3rd fluid and the cleaning liquid in 1st Embodiment It is a figure which shows the image by the micrograph of the cell fiber in one Embodiment. It is a figure which shows the flowchart of the cell fiber manufacturing method in 2nd Embodiment. It is a graph which shows the time change of the liquid feed amount of the 1st fluid, the 2nd fluid and the cleaning liquid in 2nd Embodiment. It is a schematic diagram which shows the whole structure of the cell fiber manufacturing system in 3rd Embodiment. It is a figure which shows the flowchart of the cell fiber manufacturing method in 3rd Embodiment. It is a graph which shows the time change of the liquid feed amount of the 1st fluid, the 2nd fluid and the cleaning liquid in 3rd Embodiment.

Hereinafter, embodiments will be described with reference to the drawings. In the drawings below, the same or similar parts are designated by the same or similar reference numerals.

[First Embodiment]
FIG. 1 is a schematic view showing the overall configuration of the cell fiber manufacturing system according to the first embodiment. The cell fiber manufacturing system 100 may have a first storage unit 102, a second storage unit 104, a third storage unit 106, a nozzle 200, and a reservoir 300.

The first storage unit 102 stores a first fluid containing cells. The first fluid may be, for example, a gel or liquid (suspension) containing cells. The type of these gels or liquids is not particularly limited. These gels or liquids may be, for example, chitosan gel, collagen gel, gelatin, peptide gel, fibrin gel, laminin gel, nanocellulose, purulan, dextran, medium, or alginic acid, or a mixture thereof.

The type of cells contained in the first fluid is not particularly limited. Such cells include, for example, ES cells and iPS cells having pluripotency, various stem cells having pluripotency (hemogenic stem cells, nerve stem cells, mesenchymal stem cells, etc.), and stem cells having unique differentiation (such as stem cells of mesenchymal lineage). Hepatic stem cells, reproductive stem cells, etc.) may be used. Instead of these, the cells contained in the first fluid include various differentiated cells, such as muscle cells such as skeletal muscle cells and myocardial cells, nerve cells such as cerebral cortex cells, fibroblasts, epithelial cells, and hepatocytes. It may be pancreatic β cells, skin cells, or the like. Furthermore, it should be noted that the "cells" contained in the first fluid are not limited to a single cell, but include microorganisms such as bacteria in addition to cell tissues composed of a plurality of cells.

The first fluid is a variety of growth factors suitable for cell culture, cell maintenance and proliferation, cell function expression, etc., such as epithelial growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor ( It may contain TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor (NGF) and the like.

The second storage unit 104 stores a second fluid (solution that is the source of the hydrogel) for preparing the hydrogel. The second fluid is preferably alginate or agarose. More specifically, the second fluid may be, for example, sodium alginate, potassium alginate, ammonium alginate, or a combination thereof. In addition, alginic acid may be a natural extract or a chemically modified one. Examples of the chemically modified alginic acid include methacrylate-modified alginic acid. Further, the second fluid may be a mixed system of the above-mentioned alginate and agar (Agar), agarose (Agarose), polyethylene glycol (PEG), polylactic acid (PLA), nanocellulose and the like.

The third storage unit 106 stores a third fluid that gels the second fluid when it comes into contact with the second fluid. When the above-mentioned second fluid is the above-mentioned alginate polymer material, the third fluid may be a solution containing polyvalent cations. Examples of such a solution include a solution containing calcium ions 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 inflow port for the first fluid, and fluidly communicates with the first storage unit 102. The second inlet 220 is an inflow port for the second fluid and fluidly communicates with the second reservoir 104. The third inlet 230 is an inflow port for the third fluid and fluidly communicates with the third storage unit 106.

In the present specification, the flow path from the first inlet 210 or the first storage unit 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 the present specification, the flow path from the second inlet 220 or the second storage portion 104 to the confluence with the first flow path 212 is referred to as a “second flow path”. That is, the second fluid joins the first flow path 212 through the second flow path 222 and then reaches the injection port 240. Here, the confluence of the first flow path 212 and the second flow path 222 is preferably located in the nozzle 200.

The second flow path 222 is configured to allow the second fluid for hydrogel preparation to merge around the first fluid in the first flow path 212. Thereby, the second fluid flows around the flow of the first fluid along the direction of the flow of the first fluid. That is, the second fluid surrounds the first fluid in a cross section orthogonal to the flow of the first fluid. More specifically, it is preferable that the first fluid and the second fluid flow so as to form a laminar flow.

In the present specification, the flow path from the third inlet 230 or the third storage unit 106 to the confluence with the first flow path 212 is referred to as a “third flow path”. That is, the third fluid joins the first flow path 212 through the third flow path 232 and then reaches the injection port 240. The third flow path 232 allows the third fluid to join the first flow path 212 on the downstream side of the confluence of the first flow path 212 and the second flow path 222. Here, the confluence of the first flow path 212 and the third flow path 232 is preferably located in the nozzle 200.

The third fluid flows along the flow of the second fluid around the second fluid that surrounds the first fluid on the downstream side of the confluence of the first flow path 212 and the third flow path 232. The third fluid preferably forms a laminar flow together with the first fluid and the second fluid. The second fluid gels when it comes into contact with the third fluid. Therefore, the second fluid ejected from the injection port 240 is in a partially or completely gelled state.

The sizes such as the flow path diameters of the first flow path 212, the second flow path 222, and the third flow path 232 can be appropriately designed according to the size of each part of the cell fiber to be manufactured.

It is preferable that the first flow path 212 extends in a direction generally along gravity, at least on the downstream side of the confluence of the first flow path 212 and the second flow path 222. As a result, gravity is unlikely to be applied in the direction orthogonal to the flow direction of the first fluid, so that the second fluid flowing around the first fluid is around the first fluid in a cross section orthogonal to the flow direction of the first fluid. Is easy to cover with an even thickness.

The nozzle 200 is preferably configured to be interchangeable with the cell fiber manufacturing apparatus 100. As a result, when the flow path in the nozzle 200 is clogged, the clogging can be cleared by replacing the nozzle 200. In particular, since the portion of the nozzle 200 includes a narrow flow path, there is a higher possibility of clogging than other portions of the apparatus. 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 emits a first pump 112 that sends the first fluid toward the injection port 240, a second pump 114 that sends the second fluid toward the injection port 240, and a third fluid. It may have a third pump 116 that pumps fluid towards the outlet 240. The first pump 112, the second pump 114, and the third pump 116 may be configured so that the flow rates and flow velocities of the first fluid, the second fluid, and the third fluid can be adjusted, respectively.

The flow rate settings in 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 have a control unit that controls various pumps 112, 114, 116, 132.

The cell fiber manufacturing system 100 has a first sensor 122 that detects the liquid feeding state of the first fluid, a second sensor 124 that detects the liquid feeding state of the second fluid, and a second sensor that detects the liquid feeding state of the third fluid. It may have 3 sensors 126 and. The first sensor 122, the second sensor 124, and the third sensor 126 detect whether or not the first fluid, the second fluid, and the third fluid are flowing without clogging, respectively.

The cell fiber manufacturing system 100 may have a notification unit that notifies the user of various types of information. The notification unit may be, for example, a display, a light emitting unit, a buzzer, or the like. For example, when an abnormality such as clogging is found 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 the display, a light emission or a sound indicating an abnormality, or the like.

The cell fiber manufacturing system 100 may include a reservoir 300 that receives an object ejected from an injection port 240, and a stage 400 that mounts the reservoir 300. During the production of cell fibers, the reservoir 300 preferably contains a solution suitable for receiving the cell fibers. Such a solution may be, for example, saline.

The cell fiber manufacturing system 100 preferably has an adjusting mechanism 410 that varies the distance between the reservoir 300 and the outlet 240. In this embodiment, the stage 400 is configured to be able to move up and down. Instead, the nozzle 200 may be configured to be able to move up and down.

The adjusting mechanism 410 allows the positional relationship between the ejection port 240 and the liquid level in the reservoir 300 to be freely changed during the production of the cell fiber. Here, during the production of the cell fiber, the ejection port 240 is preferably located below the liquid level in the reservoir 300, that is, in the liquid in the reservoir 300. As a result, the cell fibers ejected from the ejection 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 have a detector that can estimate or detect that the outlet 240 is located in the liquid in the reservoir 300. As a result, the adjusting mechanism 410 can automatically change the distance between the reservoir and the ejection port so that the ejection port 240 is located in the liquid in the reservoir 300. Such a detection unit may be, for example, an imaging device (for example, a camera) provided in a cell fiber manufacturing system. The image pickup device can detect the contact between the liquid level in the reservoir and the ejection port 240 of the nozzle.

The cell fiber manufacturing system 100 may have a cleaning mechanism for cleaning at least a part of the first flow path 212, the second flow path 222, and the third flow path 232. In the present embodiment, the cell fiber manufacturing system 100 may include a cleaning container 130 for storing the cleaning liquid and a cleaning pump 132 capable of sending the cleaning liquid from the cleaning container 130. The cleaning solution is preferably physiological saline.

It is preferable that the cleaning container 130 fluidly communicates with at least one of the first flow path 212, the second flow path 222, and the third flow path 232 via the cleaning flow path 134. That is, the cleaning flow path 134 can allow the cleaning liquid to flow into at least one of the first flow path 212, the second flow path 222, and the third flow path 232. As a result, at least a part of the first flow path and the second flow path can be washed through the cleaning flow path.

The cleaning pump 132 may be configured to send the cleaning liquid from the cleaning flow path 134 to the injection port 240. As a result, the cleaning liquid can flow from the cleaning flow path 134 to the injection port 240 via the first flow path 212, the second flow path 222, and / or the third flow path 232. Therefore, clogging in the flow paths 212, 222, 232 can be suppressed. In addition, there is an advantage that the flow paths 212, 222, and 232 can be washed without disassembling the cell fiber manufacturing system 100.

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

In this case, the cleaning liquid can effectively clean at least a part of the third flow path 232, particularly near the confluence of the first flow path 212 and the third flow path 232, via the cleaning flow path 134. .. The third flow path 232 is a flow path for the third fluid that gels the second fluid. If the second fluid unintentionally flows into the third flow path 232, the second fluid will gel in 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 embodiment, since the cleaning flow path 134 communicates with the third flow path 232, clogging can be suppressed or eliminated by the cleaning liquid, particularly near the confluence of the first flow path 212 and the third flow path 232. it can.

In the embodiment shown in FIG. 1, the cleaning flow path 134 communicating with the cleaning container 130 communicates with the third flow path 232. Instead, the cleaning flow path 134 communicating with the cleaning container 130 may communicate with the first flow path 212 and / or the second flow path 222. The cleaning flow path 134 may be configured so that the cleaning liquid can flow to a place where clogging of the flow path is likely to occur. Further, the cleaning flow path 134 communicating with the cleaning container 130 may be communicated not only at one of the first flow path 212, the second flow path 222, and the third flow path 232, but also at a plurality of places. ..

It is preferable that the valve 150 is provided in the cleaning flow path 134. The valve 150 may be, for example, an on-off valve or a one-way valve. As a result, the cleaning flow path 134 is closed by the valve 150, at least while the cleaning liquid is not supplied. Therefore, it is possible to prevent the first fluid, the second fluid, and / or the third fluid from flowing back into the cleaning flow path 134. In particular, in the embodiment shown in FIG. 1, it is possible to prevent the third fluid from flowing back into the cleaning flow path 134 during the non-delivery of the cleaning liquid.

As shown in FIG. 1, the valve 170 may be provided in the third flow path 232. The valve 170 may be, for example, an on-off valve or a one-way valve. As a result, even after the third pump 116 is stopped, the third fluid is prevented from flowing out to the flow path 232 and the flow path 212, and the first flow path 212 and the third flow path 232 of the third flow path 232 merge. In the vicinity of the point, clogging due to gelation of the second fluid can be suppressed. The valve 170 may be used in place of or with the cleaning mechanism described above. However, the valve 170 is not an essential configuration and may not be provided if it is unnecessary.

[Cell fiber manufacturing method]
FIG. 2 is a diagram showing a flowchart of the cell fiber manufacturing method according to the first embodiment. FIG. 3 is a graph showing the time change of the feed amount of the first fluid, the second fluid, the third fluid, and the washing liquid in the period from the start of the cell fiber production to the end of the production. It should be noted that the vertical axis of FIG. 3 shows the relative value of the liquid feed amount. The cell fiber manufacturing method in the first embodiment can be realized by using the cell fiber manufacturing system 100 described above.

First, a reservoir 300 storing a liquid such as physiological saline is placed on the stage 400 of the cell fiber manufacturing system 100, and the distance between the ejection 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 of the reservoir 300.

Here, the cell fiber manufacturing system 100 automatically estimates or detects that the ejection port 240 is located in the liquid in the reservoir 300, and the adjusting mechanism 410 automatically determines the distance between the reservoir 300 and the ejection port 240. It is preferable to fluctuate. However, the user may manually set the distance between the reservoir 300 and the ejection port 240.

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

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

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

However, it is preferable that the cleaning liquid feeding is stopped at the same time as the start of the third fluid feeding. This reduces the time required for the process of stopping the feed of the cleaning fluid and starting the feed of the third fluid.

More preferably, the feed rate of the third fluid is gradually increased while gradually lowering the feed volume of the cleaning liquid. As a result, it is possible to prevent the second fluid from entering the third flow path 232 in the process of stopping the feeding of the cleaning liquid and starting the feeding of the third fluid. Further, in this case, it is more preferable to form a laminar flow with the second fluid and the cleaning liquid, and then gradually increase the liquid feeding amount of the third fluid while gradually reducing the liquid feeding amount of the cleaning liquid. As a result, 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, the possibility that the second fluid and the third fluid are mixed by the turbulent flow in the process of starting the flow of the third fluid is reduced, and the possibility that the flow path in the nozzle 200 is clogged by the gelled second fluid is further reduced. Can be done.

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

Next, at the stage of starting the production of the cell fiber, both the second fluid and the third fluid reached at least the confluence of the third flow path 232 and the first flow path 212, and then preferably reached the injection port 240. Later, the feeding of the first fluid is started (step S14). The liquid feeding of the first fluid can be carried out by the first pump 112.

As a result, the first fluid is sent after the second fluid and the third fluid are merged. That is, after the second fluid is partially or completely gelled by the third fluid, the first fluid containing cells begins to merge with the second and third fluids. That is, the first fluid containing cells is fed after the tip portion of the cell fiber is partially or completely gelled. This makes it possible to further prevent cells from leaking from the tip of the fiber. In particular, even when the cells are cultured in the cell fiber, it is possible to prevent the cells from leaking from the tip of the cell fiber.

Next, the liquid feeds of the first fluid, the second fluid, and the third fluid are maintained for a predetermined period (step S15). During this period, the product produced by the confluence of the first fluid, the second fluid, and the third fluid is ejected from the injection port 240. Specifically, the ejected substance is a cell containing a substantially tubular hydrogel generated by the confluence of the second fluid and the third fluid, and a first fluid containing cells filled in the hydrogel. It is a fiber. The length of the cell fiber can be adjusted according to the length of the liquid feeding period in step S15.

At the stage of stopping the production of the cell fiber, first, the feeding of the first fluid is stopped (step S16). Even after the liquid feeding of the first fluid is stopped, it is preferable to continue the liquid feeding of the second fluid and the third fluid for a predetermined period. This ensures that the rear end portion of the cell fiber is sufficiently closed with the (gelled) second fluid. This makes it possible to prevent cells in the cell fiber from leaking from the rear end of the fiber.

Next, at the stage of stopping the production of the cell fiber, the feeding of the third fluid is stopped and the feeding of the washing liquid is started (step S17). It is preferable that the cleaning liquid feeding is stopped at the same time as the start of the third fluid feeding. As a result, the fluid flowing in the nozzle 200 is gradually replaced with the cleaning liquid from the third fluid. As a result, the third fluid in the nozzle 200 is pushed out from the injection port 240 by the cleaning liquid. As a result, the second fluid does not come into contact with the third fluid, gelation of the second fluid is suppressed, and the occurrence of clogging at the stop stage can be suppressed.

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

Preferably, the stoppage of the cleaning liquid delivery is simultaneous with or after the stoppage of the second fluid delivery. As a result, it is possible to prevent the second fluid from flowing back into the third flow path 232.

In the method described above, the feeding of the second fluid is stopped after the feeding of the third fluid is stopped at the stage of stopping the production of the cell fiber. In this case, there is a period in which the second fluid flows even after the third fluid that gels the second fluid is stopped. Therefore, there is a period during which the second fluid pushes the residue of the third fluid out of the outlet 240. Therefore, it is possible to prevent the gelled second fluid from staying in the nozzle 200 and causing clogging after the production of the cell fiber is stopped.

After steps S11-S18 described above, the cell fibers produced in the reservoir 300 may be replaced in the culture medium. In this case, since the cells are confined in the cell fiber, the cells can be easily transferred into the culture medium. In addition, since cells are hardly mixed in the waste liquid in the reservoir 300, the waste liquid can be easily treated.

The cells in the cell fiber may be cultured for a predetermined period in a state of being transferred to the culture medium. Since both ends of the fiber in the extending direction are closed with hydrogel, the cells remain confined in the cell fiber even when the cells increase.

Whether the cleaning solution, the second fluid and / or the third fluid has reached an arbitrary position in the flow path or the injection port 240 at the manufacturing start stage or the stop stage of the cell fiber described above is determined by the human eye or the cell fiber. It can be judged artificially or automatically by the manufacturing system. For example, a camera is mounted on a cell fiber manufacturing system, and the cell fiber manufacturing system automatically determines whether or not a cleaning liquid, a second fluid, and / or a third fluid is ejected from an injection port 240. 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 to allow the cleaning fluid, the second fluid, and / or the third fluid to flow in any flow path. It may be determined whether the position or the ejection port 240 has been reached. In this case, the cell fiber manufacturing system is based on the distance between the sensors 122, 124, 126 and any position or outlet 240 in the flow path and the flow velocity of the various fluids. The time from the sensors 122, 124, 126 to reach an arbitrary position in the flow path or the injection port 240 may be estimated. As a result, the cell fiber manufacturing system uses the first sensor 122, the second sensor 124, and the third sensor 126 to allow the cleaning liquid, the second fluid, and / or the third fluid to shoot at an arbitrary position or emission in the flow path. It can be automatically determined whether or not the exit 240 has been reached.

FIG. 4 is a diagram showing a micrograph image of a cell fiber in one embodiment. FIG. 4 is a photomicrograph of a cell fiber produced by the above-mentioned apparatus and method. FIG. 4 shows the state after the cells have been cultured in the cell fiber for a long period of time.

The cell fiber includes a plurality of cells 800 arranged along the fiber and a hydrogel 810 that wraps around the cells in a cross section intersecting the extending direction of the fiber. The hydrogel 810 is closed at both ends 820 in the extending direction of the fiber so as to confine the cells. The hydrogel 810 is composed of a 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. Further, in the example shown in FIG. 4, the cells in the first fluid are cardiomyocytes derived from iPS cells.

The outer diameter of the cell fiber is not particularly limited, but may be in the range of, for example, about 1 μm to 5 mm. The length of the cell fiber 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 be in the range of, for example, about 100 nm to 1000 μm. Further, at both ends in the extending direction of the cell fiber, the length of the region of the gel portion containing no cells is preferably, for example, 10 times or more the outer shape 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 containing no cells may be larger than 1 mm, preferably 5 mm, and more preferably 20 mm. This makes it possible to prevent cells in the fiber from leaking from the end of the fiber.

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

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

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

By flowing the cleaning liquid before sending 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 flow path in the nozzle 200, the second fluid may gel and cause clogging. By sending the cleaning liquid first, the possibility of such clogging can be reduced.

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

Next, the second fluid for hydrogel preparation is sent toward the injection port 240 (step S34). The liquid feeding of the second fluid can be carried out by the second pump 114. The delivery of the second fluid is started after the third fluid has reached 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, the delivery of the second fluid is started after the flow of the third fluid is stable. In step S34, it is preferable that a laminar flow is formed by the second fluid and the third fluid.

Next, at the stage of starting the production of the cell fiber, both the second fluid and the third fluid reached at least the confluence of the third flow path 232 and the first flow path 212, and then preferably reached the injection port 240. Later, the feeding of the first fluid is started (step S35). The liquid feeding of the first fluid can be carried out by the first pump 112.

As a result, the first fluid is sent after the second fluid and the third fluid are merged. That is, after the second fluid is partially or completely gelled by the third fluid, the first fluid containing cells begins to merge with the second and third fluids.

Next, the liquid feeds of the first fluid, the second fluid, and the third fluid are maintained for a predetermined period (step S36). Step S36 is the same as step S15 in the first embodiment.

Next, in the stage of stopping the production of the cell fiber, first, the feeding of the first fluid is stopped (step S37). Even after the liquid feeding of the first fluid is stopped, it is preferable to continue the liquid feeding of the second fluid and the third fluid for a predetermined period. This ensures that the rear end portion of the cell fiber is sufficiently closed with the (gelled) second fluid. This makes it possible to prevent cells in the cell fiber from leaking from the rear end of the fiber.

Next, at the stage of stopping the production of the cell fiber, the feeding of the second fluid is stopped (step S38). Next, the feeding of the third fluid is stopped (step S39). Alternatively, the liquid feeding of the third fluid may be stopped and then the liquid feeding of the second fluid may be 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 prevent the residue from remaining in the flow path in the nozzle 200.

In the second embodiment, at the production start stage, the liquid feeding of the third fluid is started, and then the liquid feeding of the second fluid is started. Instead, at the production start stage, the second fluid and the third fluid may start the liquid feeding at substantially the same timing.

[Third Embodiment]
FIG. 7 is a schematic view showing the overall configuration of the cell fiber manufacturing system according to the third embodiment. In FIG. 7, the same reference numerals are given to the same configurations as those in the first embodiment. Further, the description of the configuration similar to that of the first embodiment may be omitted.

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

In the third embodiment, the cell fiber manufacturing system 100 does not include the third reservoir 106 or the third flow path 232. The third fluid, which gels the second fluid, is placed in the reservoir 300 at the stage of manufacturing the cell fiber. The material of the third fluid may be the same as that of the first embodiment.

In the third embodiment, the injection port 240 ejects the first fluid and the second fluid together into the third fluid in the reservoir 300. As a result, the second fluid comes into contact with the third fluid in the reservoir 300 and is gelled.

The cell fiber manufacturing system 100 may have a cleaning mechanism for cleaning at least a part of the first flow path 212 and the second flow path 222. In the third embodiment, the cleaning flow path 134 is configured to communicate with the second flow path 222 and allow the cleaning liquid to flow into the second flow path 222. Instead, the cleaning flow path 134 may be configured to communicate with the first flow path 212 and allow the cleaning liquid to flow into the first flow path 212.

[Cell fiber manufacturing method]
FIG. 8 is a diagram showing a flowchart of the cell fiber manufacturing method according to the third embodiment. FIG. 9 is a graph showing the time change of the amount of the first fluid, the second fluid, and the washing liquid to be fed during the period from the start of the cell fiber production to the end of the production. It should be noted that the vertical axis of FIG. 9 shows the relative value of the liquid feed amount. The cell fiber manufacturing method in the third embodiment can be realized by using the cell fiber manufacturing system 100 shown in FIG. 7.

First, a reservoir 300 storing a liquid such as physiological saline is placed on the stage 400 of the cell fiber manufacturing system 100, and the distance between the ejection 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 ejection port 240 is located in the third fluid in the reservoir 300, and the adjusting mechanism 410 automatically between the reservoir 300 and the ejection port 240. It is preferable to change the distance. However, the user may manually set the distance between the reservoir 300 and the ejection port 240.

Next, at the stage of starting the production of the cell fiber, the 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 of the first flow path and the second flow path, and preferably reaches the injection port 240, the liquid feeding of the first fluid is started (step S24). .. At this time, it is preferable to form a laminar flow with the first fluid and the second fluid. Since the first fluid containing the cells is delivered after the second fluid, the tip of the cell fiber is sufficiently closed by the (gelled) second fluid. This makes it possible to prevent cells from leaking from the tip of the fiber during the production of the cell fiber.

Next, the liquid feeds of the first fluid and the second fluid are maintained for a predetermined period (step S25). During this period, the confluence of the first fluid and the second fluid is ejected from the injection port 240. The second fluid comes into contact with the third fluid in the reservoir 300 and gels. As a result, a cell fiber containing a substantially tubular hydrogel and a first fluid containing cells filled in the hydrogel is produced. The length of the cell fiber can be adjusted according to the length of the liquid feeding period in step S25.

At the stage of stopping the production of the cell fiber, first, the feeding of the first fluid is stopped (step S26). Even after the liquid feeding of the first fluid is stopped, it is preferable to continue the liquid feeding of the second fluid for a predetermined period. This ensures that the rear end portion of the cell fiber is sufficiently closed with the (gelled) second fluid. Therefore, it is possible to prevent cells in the cell fiber from leaking from the rear end of the fiber.

Next, at the stage of stopping the production of the cell fiber, the feeding of the second fluid is stopped and the feeding of the washing liquid is started (step S27). As a result, the inside of the nozzle 200 can be washed with a washing liquid at the stage of stopping the production of the cell fiber.

Next, at the stage of stopping the production of the cell fiber, the feeding of the washing liquid is stopped (step S28). Similar to the first embodiment, after steps S21 to S28 described above, the cell fibers generated in the reservoir 300 may be replaced in the culture medium.

As mentioned above, although the content of the invention has been disclosed through embodiments, the statements and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure reveals to those skilled in the art various alternative embodiments, examples and operational techniques. Therefore, the technical scope of the present invention is defined only by the matters specifying the invention relating to the reasonable claims from the above description.

For example, the cell fiber manufacturing method in the first embodiment, the second embodiment, and the third embodiment described above includes a step of feeding a washing liquid at a start stage or a stop stage. However, the step of sending the cleaning liquid is not an indispensable step, and may be performed as needed. Specifically, the step of delivering the cleaning liquid may not be carried out in one or both of the production start stage and the stop stage. Even when cleaning is not performed in one or both of the production start stage and the stop stage, the order of flowing the first fluid, the second fluid, and / or the third fluid may be as described above. The cell fiber manufacturing system is provided with the above-mentioned cleaning flow path 134 and the like so that cleaning can be performed at a timely timing such as maintenance even when there is no step of sending the cleaning liquid at the production start stage and the production stop stage. Is preferable.

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

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

It should be noted that a program for causing a computer to execute the above-mentioned cell fiber manufacturing method described with reference to the flowcharts shown in FIGS. 2, 5 and 8 is also included in the scope of the present invention. Such computer programs can be stored on various types of non-transitory computer readable media and supplied to the computer. Non-transient computer-readable media include various types of tangible storage media. Examples of non-temporary 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, It includes a CD-R / W, a semiconductor memory (for example, a mask ROM, a PROM (Programmable ROM), an EPROM (Erasable PROM), a flash ROM, and a RAM (random access memory)). The program may also be supplied to the computer by various types of temporary computer readable media. Examples of temporary computer-readable media include electrical, optical, and electromagnetic waves. The temporary computer-readable medium can supply the program to the computer via a wired communication path such as an electric wire and an optical fiber, or a wireless communication path.

From the above-described embodiment, it can be understood that at least the following inventions are also explained.

In the cell fiber manufacturing system according to one aspect, a first flow path through which a first fluid containing cells flows and a second fluid for preparing a hydrogel are merged around the first fluid in the first flow path. It has two flow paths and at least an injection port for discharging the first fluid and the second fluid together. The cell fiber manufacturing system and / or method is such that the first fluid reaches at least the confluence of the first flow path and the second flow path at the start stage of cell fiber manufacturing. It is configured to be fed. Preferably, the cell fiber manufacturing system and / or method is configured such that the first fluid is delivered after the second fluid reaches the outlet at the start of cell fiber manufacturing.

Since the first fluid containing the cells is delivered after the second fluid, the tip of the cell fiber is sufficiently closed by the (gelled) second fluid. This makes it possible to prevent cells from leaking from the tip of the fiber during the production of the cell fiber. When a cell leaks from a cell fiber, it is not always easy to find out where the cell leaked. It should be noted that the present inventor has discovered that cells leak from the front end portion or the rear end portion of the cell fiber depending on the conditions, leading to the present invention.

The cell fiber manufacturing system and / or method according to another aspect is configured to stop the feeding of the second fluid after stopping the feeding of the first fluid at the stage of stopping the manufacturing of the cell fiber.

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

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

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

According to another aspect, the cell fiber manufacturing system and / or method is such that at the start stage of cell fiber manufacturing, both the second fluid and the third fluid are at least the third flow path and the first flow path. The first fluid is configured to be fed after reaching the confluence of. Preferably, the cell fiber manufacturing system and / or method is such that the first fluid is delivered after both the second fluid and the third fluid have reached the outlet at the cell fiber manufacturing initiation stage. It is configured as.

In this aspect, the first fluid is sent after the second fluid and the third fluid have merged. That is, the first fluid containing cells is fed after the tip portion of the cell fiber is partially or completely gelled. Therefore, it is possible to further prevent cells from leaking from the tip of the fiber during the production of the cell fiber.

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

In this embodiment, since the flow of the second fluid is stabilized before the third fluid flows, it becomes easy to form a laminar flow between the second fluid and the third fluid. As a result, the possibility that the second fluid and the third fluid are mixed due to the turbulent flow is reduced, and the possibility that the flow path in the nozzle 200 is clogged by the gelled second fluid can be reduced.

According to another aspect, the cell fiber manufacturing system and / or method performs the feeding of the second fluid and the third fluid after stopping the feeding of the first fluid at the stage of stopping the manufacturing of the cell fiber. It is configured to stop.

In this embodiment, the second fluid and the third fluid are merged and ejected even when the feeding of the first fluid containing the cells is stopped. That is, the leading end portion of the cell fiber is partially or completely gelled, and the trailing end portion is closed by the hydrogel. Therefore, it is possible to further prevent cells from leaking from the rear end of the fiber.

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

In this aspect, there is a period during which the second fluid flows even after the third fluid that gels the second fluid is stopped. Therefore, the second fluid pushes the residue of the third fluid out of the ejection port. Therefore, it is possible to prevent the gelled fluid from staying in the flow path and causing clogging after the production of the cell fiber is stopped.

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

In this aspect, at least a part of the first flow path and the second flow path can be washed through the washing flow path. Therefore, clogging of the flow path can be suppressed. In addition, there is an advantage 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 has a cleaning flow path that allows the cleaning liquid to flow into the third flow path.

In this aspect, at least a part of the third flow path can be washed through the washing flow path. The third flow path is a flow path for the 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. By communicating the cleaning flow path with the third flow path, it is possible to eliminate such clogging in the third flow path by the cleaning liquid.

According to another aspect, the cell fiber manufacturing system and / or method comprises a liquid delivery pump that feeds the cleaning fluid from the cleaning flow path to the ejection port.

In this aspect, the cleaning liquid can be flowed from the cleaning flow path to the injection port via the first flow path, the second flow path, and / or the third flow path by the liquid feeding pump. Further, since the third fluid gels the second fluid, clogging is likely to occur in the flow path through which the third fluid passes. Therefore, it is more preferable that the cleaning liquid passes 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 wash solution at the start of cell fiber manufacturing, at least before the start of feeding the first fluid. ..

In this aspect, the production of cell fibers can be started after cleaning the flow path in 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 at the stage of stopping the production of the cell fiber.

In this aspect, since the flow path in the nozzle can be cleaned at the stage of stopping the production of the cell fiber, it is possible to prevent clogging in the flow path after the stop.

According to another aspect, the cell fiber manufacturing system and / or method is configured to deliver the cleaning solution at the start of the cell fiber production, at least at the start or before the start of the delivery of the third fluid. Has been done.

In this aspect, the second fluid can begin to gel after cleaning the flow path in the nozzle. That is, the flow path in the nozzle can be cleaned before forming the hydrogel.

According to another aspect, the cell fiber manufacturing system and / or method is configured to feed the washing liquid at least during or after stopping the feeding of the third fluid at the stage of stopping the production of the cell fiber. ing.

In this aspect, it is possible to prevent the second fluid from flowing back into the third flow path due to the delivery of the cleaning liquid at the production stop stage. Further, even if the second fluid is mixed in the third flow path and gelled in the third flow path, it can be pushed out from the injection port by sending the cleaning liquid. As a result, clogging in the nozzle 200 can be further suppressed.

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

In this embodiment, the cleaning flow path is closed by the valve at least while the cleaning liquid is not fed, so that it is possible to prevent the first fluid, the second fluid, and / or the third fluid from flowing back into the cleaning flow path. ..

According to another aspect, the cell fiber manufacturing system and / or method has 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. As a result, clogging due to gelation of the second fluid can be suppressed in the vicinity of the confluence of the first flow path and the third flow path in the third flow path.

According to another aspect, the cell fiber manufacturing system and / or method has a reservoir that receives material ejected from the outlet and an adjusting mechanism that varies the distance between the reservoir and the outlet. ..

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

According to another aspect, the cell fiber manufacturing system and / or method has a detector that can estimate or detect that the outlet is located in the liquid in the reservoir.

In this aspect, it is possible to detect that the outlet is located in the liquid in the reservoir prior to the manufacture of the cell fiber. As a result, the adjusting mechanism can automatically change the distance between the reservoir and the ejection port so that the ejection port is located in the liquid in the reservoir.

According to another aspect, the cell fiber manufacturing system and / or method has a nozzle that includes the first flow path, the second flow path, the third flow path, and the injection port, and the nozzle is replaceable. It is configured in.

In this aspect, when the flow path in the nozzle 200 is clogged, the clogging can be easily cleared by replacing the nozzle. Therefore, the interchangeable nozzles allow the cell fiber manufacturing system to be used for a long period of time.

According to another aspect, the nozzle for the cell fiber manufacturing system is a first flow path through which a first fluid containing cells flows, and a second fluid for preparing a hydrogel, the first fluid in the first flow path. The second flow path that merges around the water and the third fluid that gels the second fluid are placed downstream of the confluence of the first flow path and the second flow path, and the first flow path is located. It has a third flow path that merges with the water, and at least an injection port that discharges the first fluid, the second fluid, and the third fluid together. The nozzle is configured to be interchangeable with respect to the cell fiber manufacturing system.

According to another aspect, the cell fiber comprises a plurality of cells aligned along the fiber and a hydrogel that wraps around the cells in a cross section that intersects the extending direction of the fiber. The hydrogel is closed at both ends in the extending direction of the fiber so as to confine the cells.

In this embodiment, the cells are closed by hydrogels at the front and back ends of the fiber. Therefore, even if the cells are cultured in the fiber, it is possible to prevent the cells from leaking from the front end or the rear end of the fiber during the production of the cell fiber.

100 Cell manufacturing system 102 1st storage unit 104 2nd storage unit 106 3rd storage unit 130 Cleaning container 132 Cleaning pump 134 Cleaning flow path 200 Nozzle 210 1st inlet 212 1st flow path 220 2nd inlet 222 2nd flow path 230 3rd inlet 232 3rd flow path 240 Outlet 300 Reservoir 400 Stage

Claims (6)

A first flow path through which a first fluid containing cells flows, and
A second flow path for merging the second fluid for hydrogel preparation around the first fluid in the first flow path,
At least an injection port that discharges the first fluid and the second fluid together ,
A fiber having a control unit that controls the first fluid to start merging with the second fluid after the second fluid merges with the third fluid that gels the second fluid at the manufacturing start stage. Manufacturing system. The fiber manufacturing according to claim 1, further comprising a third flow path for merging the third fluid into the first flow path on the downstream side of the confluence of the first flow path and the second flow path. system. The fiber manufacturing system according to claim 1, further comprising a reservoir for storing the third fluid. The control unit is configured to stop the feeding of the second fluid after stopping the feeding of the first fluid at the production stop stage, according to any one of claims 1 to 3. Cell fiber manufacturing system. A first flow path through which a first fluid containing cells flows, and
A second flow path for merging the second fluid for hydrogel preparation around the first fluid in the first flow path,
A fiber manufacturing method using an apparatus having at least an injection port for discharging the first fluid and the second fluid together.
The first fluid and the second fluid so that the first fluid begins to merge with the second fluid after the second fluid merges with the third fluid that gels the second fluid at the manufacturing start stage. A fiber manufacturing method that feeds fluid. A program for causing a computer to execute the fiber manufacturing method according to claim 5.

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