JP6939643B2 - Cell division device and cell culture device - Google Patents

Description

The present invention relates to a cell division device and a cell culture device.

Patent Documents 1 to 3 disclose an apparatus for culturing cells. For example, the cell culture apparatus disclosed in Patent Documents 1 and 2 has a cell culture tank in which the horizontal cross-sectional area gradually increases upward. According to this cell culture tank, the cell mass can be uniformly distributed in the tank according to the size of the cell mass.

Japanese Unexamined Patent Publication No. 2016-86791 Japanese Unexamined Patent Publication No. 2005-348672 International Publication No. 2014/136581

The cell culture tank is a tank for so-called suspension culture. In the cell culture tank, cells are cultured in a state in which the cells are suspended in a medium held inside the cell culture tank. In cells, a plurality of cells gather to form a cell mass, and when the cells are cultured, the cell mass grows. Inside the cell culture tank, the medium flows from bottom to top. Then, the cell mass floats at a position where the sedimentation rate according to the size of the cell mass and the ascending rate component of the medium are balanced. In culturing such cells, it is desired to keep the size of the cell mass within a desired range.

Therefore, the present invention provides a cell division device and a cell culture device capable of keeping the size of a cell mass within a desired range.

One embodiment of the present invention is a cell division device that receives a medium containing a cell mass cultured in a cell culture tank and divides the cell mass so as to be smaller than a predetermined size, and forms a flow path through which the medium flows. A tubular tube portion having an opening for flowing out the medium from the flow path and a dividing portion provided in the opening for dividing the cell mass flowing out from the opening together with the medium are provided, and the dividing portion is a tube. Includes split fiber portions that are formed around the axis of the portion and are arranged across the opening such that at least two outflow regions of equal area are formed in the opening.

In this cell division device, a division fiber portion is provided at an opening through which a medium containing a cell mass flows out. The split fiber portions are provided so as to divide the opening into at least two outflow regions, and the outflow regions are formed at equal intervals around the axis of the pipe portion. Then, the cell mass smaller than the outflow region flows out from the outflow region together with the medium. On the other hand, a cell mass larger than the outflow region is divided by the split fiber portion. Then, the cell mass contained in the medium spilled from the opening has the same size as the spill region or smaller than the spill region when viewed from the spill direction. Therefore, the size of the cell mass can be kept within a desired range.

In the cell division device according to one form, the opening may be provided at the tip of the tube portion. According to this configuration, the flow of the medium can be used as a force for pressing the cell mass against the split fiber portion. Therefore, the cell mass can be suitably divided.

In the cell dividing device according to one form, the divided fiber portion has a first fiber thread and a second fiber thread arranged in the opening so that four outflow regions are formed in the opening, and the first The fibrous yarn may intersect the second fibrous yarn. According to this configuration, four outflow regions can be formed in the opening.

In the cell dividing device according to one form, the position where the first fiber thread intersects with the second fiber thread may be on the axis of the tube portion. According to this configuration, the first fiber thread and the second fiber thread intersect on the axis of the pipe portion, that is, at the center of the opening. Therefore, it is possible to reduce the bias of the opportunity for the cell mass and the first and second fiber threads to come into contact with each other. As a result, the size of the cell mass after division can be made uniform.

In the cell division device according to one form, the tube portion has a groove-shaped fiber arrangement portion provided on the end face surrounding the opening, and the division fiber portion may be arranged in the fiber arrangement portion. According to this configuration, the position of the split fiber portion with respect to the end face is reliably maintained even when the split fiber portion is pressed by the medium and the cell mass. As a result, the relationship between the outflow regions is maintained, so that the size of the cell mass after division can be maintained.

The cell culture apparatus, which is another form of the present invention, is connected to a cell culture tank for culturing a cell mass contained in the medium and a cell culture tank, receives the medium from the cell culture tank, and causes the cell mass to have a predetermined size. A tubular portion having an opening for forming a flow path through which the medium flows and allowing the medium to flow out from the flow path, and a divided portion provided in the opening. , A division that divides the cell mass that flows out of the opening together with the medium, the division is formed around the axis of the tube, and at least two outflow regions having equal areas with each other are formed in the opening. Includes split fiber portions arranged across the opening so as to. This cell culture device includes the above-mentioned cell division device. Therefore, the cell culture apparatus can culture the cell mass while keeping the cell mass at a desired size.

According to the present invention, there is provided a cell division device and a cell culture device capable of keeping the size of a cell mass within a desired range.

FIG. 1 is a diagram showing a configuration of a cell culture device including a cell division device according to the present embodiment. FIG. 2 is a perspective view showing a main part of the cell division device shown in FIG. FIG. 3 is an end view of the opening of the cell division device in a plan view. Part (a) of FIG. 4 is a plan view of the opening of the cell dividing device according to the first modification, and part (b) of FIG. 4 is a plan view of the opening of the cell dividing device according to the second modification. It is an end view.

Hereinafter, one form of the present disclosure will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are designated by the same reference numerals, and duplicate description will be omitted.

As shown in FIG. 1, the cell culture device 1 having an airtight structure includes a cell culture tank 2, an aeration tank 3, a waste liquid tank 4, and a cell division device 5. The cell culture tank 2 is connected to the aeration tank 3 by the medium outflow pipe 6 and the medium introduction pipe 7. Further, the aeration tank 3 is connected to the waste liquid tank 4 by the medium discharge pipe 8.

In the cell culture tank 2, the cells to be cultured are cultured in the medium M. The cell culture tank 2 contains the medium M. Medium M contains cell mass S. The medium M may contain cells or a carrier to which the cells are attached, in addition to the cell mass S. The carrier is a fine particle, also called a microphone carrier. The carrier serves as an attachment base for cells. The cells then proliferate on the carrier.

The upper part of the cell culture tank 2 is opened, and the bottom of the cell culture tank 2 is closed. The cell culture tank 2 receives the medium M from the lower part and flows the medium M toward the upper part. This flow of medium M from the lower part to the upper part is called a plug flow. The plug flow is a flow having a rotation speed component that rotates around the axis of the cell culture tank 2 and an ascending speed component that goes from the lower part to the upper part.

An airtight lid 9 is provided on the upper part of the cell culture tank 2. An opening is formed at the upper end of the cell culture tank 2, and the opening is hermetically closed by the lid 9. The lid 9 is connected to the aeration tank 3 by the medium outflow pipe 6. One end of the medium outflow pipe 6 communicates with the lowermost position on the bottom surface of the lid 9. The other end of the medium outflow pipe 6 is connected to the aeration tank 3.

The aeration tank 3 stores the medium M to be supplied to the cell culture tank 2 and adjusts the gas concentration of the medium M. The other end of the culture medium introduction tube 7 is connected to the aeration tank 3. The medium introduction tube 7 is provided with a pump 11 for supplying the medium M from the aeration tank 3 to the cell culture tank 2. A gas supply pipe 12 and a medium supply pipe 13 are further connected to the aeration tank 3.

The waste liquid tank 4 stores the excess medium M discharged from the aeration tank 3. One end of the culture medium discharge pipe 8 is connected to the waste liquid tank 4. The position of one end of the medium discharge pipe 8 is above the liquid level of the waste liquid stored in the waste liquid tank 4.

Next, the operation of the cell culture device 1 will be described. In the present embodiment, the cell mass S is suspended in the medium M, and the cells constituting the cell mass S are proliferated.

First, a new medium M is supplied to the aeration tank 3 from the medium supply pipe 13. The supplied medium M is stored in the aeration tank 3. Further, gas such as oxygen and carbon dioxide sterilized by the air filter 14 is supplied to the aeration tank 3 via the gas supply pipe 12. The gas supplied by the gas supply pipe 12 adjusts the oxygen concentration and hydrogen ion concentration (pH) of the medium M stored in the aeration tank 3 to conditions suitable for aerobic culture of cell culture. The condition-adjusted medium M is supplied from the aeration tank 3 to the cell culture tank 2 via the medium introduction tube 7 by the pump 11.

As the cell culture progresses, in the medium M, medium components such as oxygen decrease, while waste products excreted from the cells increase. The medium M is guided from the upper end of the cell culture tank 2 to the lid 9 and flows out to the aeration tank 3 via the medium outflow pipe 6. The medium M flowing out to the aeration tank 3 is again supplied with a medium component such as oxygen in the aeration tank 3. Then, it is supplied from the aeration tank 3 to the cell culture tank 2 via the medium introduction tube 7. On the other hand, when a new medium M is added from the medium supply pipe 13, the surplus medium M due to the addition of the medium M is discharged to the waste liquid tank 4 via the medium discharge pipe 8.

Therefore, the cell mass S floats at a position where the magnitude of the ascending velocity component of the plug flow and the sedimentation velocity are balanced. For example, a cell mass Sa having a relatively small particle size has a low sedimentation rate. Therefore, it floats in the upper layer where the rising velocity component is small. Then, the cell mass S grows larger as the cells are cultured in the upper layer where the flow state is gentle. The enlarged cell mass Sb floats in the lower layer where the velocity of the ascending flow and the velocity of the sedimentation are balanced.

<Cell division device>
The cell division device 5 receives the medium M containing the cell mass S from the cell culture tank 2 and divides the cell mass S larger than a predetermined size. The divided cell mass S is excreted together with the medium M. Further, the divided cell mass S may be returned to the cell culture tank 2 again together with the medium M. The cell division device 5 is connected to the cell culture tank 2 via the medium supply tube 16. A pump 17 may be provided in the medium supply pipe 16. Further, the cell division device 5 discharges the medium M containing the cell mass S divided through the medium discharge tube 18. The cell division device 5 may be connected to the cell culture tank 2 via the medium recovery tube 19.

The cell dividing device 5 has a front accommodating portion 21, a rear accommodating portion 22, and a cylindrical dividing nozzle 23 (tube portion) as main components.

The pre-accommodation unit 21 is connected to the medium supply pipe 16 and temporarily holds the medium M received from the cell culture tank 2. A predetermined static pressure may be applied to the medium M contained in the pre-containment unit 21 by a pump 17 or a compressor. Then, the split nozzle 23 is connected to the front storage section 21, and the medium M is provided from the front storage section 21 to the split nozzle 23.

The rear accommodating portion 22 is connected to the split nozzle 23 and temporarily holds the medium M flowing out of the split nozzle 23. Then, the medium discharge tube 18 is connected to the rear accommodating portion 22, and the medium M containing the cell mass divided from the rear accommodating portion 22 is discharged. A medium recovery tube 19 may be connected to the post-containment unit 22, and the medium M containing the cell mass S divided from the post-containment unit 22 may be returned to the cell culture tank 2 again.

The front accommodating portion 21 and the rear accommodating portion 22 may be provided as needed, and may be omitted as appropriate depending on the configuration of the cell culture tank 2. For example, the cell division device 5 may not have the front accommodating portion 21, and the medium supply tube 16 may be directly connected to the division nozzle 23. Further, the cell dividing device 5 may not have the rear accommodating portion 22, and the dividing nozzle 23 may be directly connected to the medium discharge tube 18 or the medium recovery tube 19.

As shown in FIG. 2, the division nozzle 23 divides the cell mass S contained in the medium M received from the front storage unit 21, and provides the medium M containing the divided cell mass S to the rear storage unit 22.

The split nozzle 23 has a substantially tubular shape, and the base end is watertightly connected to the front accommodating portion 21, and the tip end 23a is watertightly connected to the rear accommodating portion 22. The base end of the dividing nozzle 23 receives the medium M, and the tip portion 23a of the dividing nozzle 23 discharges the medium M. The split nozzle 23 forms a flow path 24 inside the split nozzle 23. The medium M flows from the proximal end side toward the distal end portion 23a in the flow path 24. The split nozzle 23 shown in FIG. 2 has a tapered shape in which the diameter of the flow path 24 gradually decreases in the direction D1 from the base end toward the tip end portion 23a. The diameter of the flow path 24 may be constant from the base end to the tip end portion 23a.

The area of the circular opening at the proximal end may be slightly larger than, for example, the external dimensions of the cell mass S (for example, 400 μm or more and 500 μm or less). The external dimension of the cell mass S referred to here is the projected area when the cell mass S is viewed from the direction of the axis A23 of the dividing nozzle 23. Further, the area of the circular opening at the tip portion 23a may be, for example, about the same as the outer dimension of the cell mass S. That is, in the flow path of the split nozzle 23, one cell mass S having an average size may be present in the cross section thereof. Further, if the cell mass S has a size smaller than the average size (for example, 400 μm or less), one or more cell masses may be present in the cross section.

The medium M flows out from the opening region F (opening) formed in the tip portion 23a of the split nozzle 23. This opening region F is a region surrounded by the end surface 23c of the split nozzle 23. The opening region F includes four outflow regions F1, F2, F3, and F4 separated from each other. These outflow regions F1, F2, F3, and F4 are separated by a dividing portion 26. More specifically, it is separated by two fiber threads 27 and 28 (divided fiber portions) included in the split portion 26. The fiber threads 27 and 28 are resin or metal threads, for example, the resin thread is a PEEK fiber having a diameter of 25 micrometer, and the metal thread has a diameter of 25 micrometer. Wires made of stainless steel, titanium, etc.

The fiber thread 27 (first fiber thread) is arranged along the radial direction R1 of the circular opening region F. That is, the fiber thread 27 passes through the center so as to cross the opening region F, and divides the opening region F into two. A plurality of groove-shaped thread arranging portions 29 (fiber arranging portions) are provided on the end surface 23c of the split nozzle 23, and the fiber threads 27 are fitted into the thread arranging portions 29. The pair of thread arranging portions 29 face each other with the center of the opening region F interposed therebetween. The end of the fiber thread 27 fitted in the thread arranging portion 29 extends along the outer peripheral surface 23b of the split nozzle 23. Then, for example, the fiber thread 27 is held by the band 31 provided so as to tighten the outer peripheral surface 23b of the split nozzle 23.

As shown in FIG. 3, the fiber thread 28 (second fiber thread) is arranged along the radial direction R2 of the circular opening region F. At this time, the fiber thread 28 is arranged in a direction orthogonal to the fiber thread 27. That is, the position where the fiber thread 28 intersects the fiber thread 27 is on the axis A23 of the split nozzle 23. Further, the fiber threads 27 and 28 may or may not be fixed to each other at points where they intersect each other. The angle K formed by the fiber thread 28 and the fiber thread 27 is 90 degrees. That is, the fiber threads 27 and 28 divide the opening region F into four equal parts around the axis A23. Then, for example, the area of the outflow region F1 is 1/4 of the opening region F. Similarly, the areas of the outflow regions F2, F3, and F4 are also 1/4 of the opening region F. That is, the areas of the outflow regions F1, F2, F3, and F4 are substantially equal to each other. For example, in the split nozzle 23, the area of one outflow region does not become more than twice as large as the area of another outflow region.

As shown in FIG. 2 again, the medium M containing the cell mass S flows into the split nozzle 23. The cell mass S has an average size and moves toward the tip side while one is present in the cross section. Then, one cell mass S is caught in the fiber threads 27 and 28.
Since pressure is applied to the cell mass S by the medium M from the front accommodating portion 21 side, the cell mass S is pressed toward the fiber threads 27 and 28. Then, the portions of the cell mass S in contact with the fiber threads 27 and 28 are divided, and finally divided into four cell masses S1, S2, S3, and S4. When the diameter of the cell mass S is smaller than the inner diameter of the tube of the split nozzle 23 and is a diameter that allows passage, two cells that pass near the center of the cell mass S in contact with the fiber threads 27 and 28. The portion that can be divided by a line and does not come into contact with the fiber threads 27 and 28 is less likely to be damaged.

That is, the sizes of the outflow regions F1, F2, F3, and F4 correspond to the sizes of the desired cell masses S1, S2, S3, and S4. Therefore, the medium M flowing out from the dividing nozzle 23 includes cell clusters S1, S2, S3, S4 having the same size as the outflow regions F1, F2, F3, and F4, or smaller cell clusters. Therefore, the size of the cell mass S falls within a desired range.

In short, in this cell division device 5, the division portion 26 is provided in the opening region F from which the medium M containing the cell mass S flows out. The dividing portion 26 is provided so as to divide the opening region F into four outflow regions F1, F2, F3, and F4. The outflow regions F1, F2, F3, and F4 are formed at equal intervals around the axis A23 of the split nozzle 23. Then, the cell mass S smaller than the outflow regions F1, F2, F3, and F4 flows out from the outflow regions F1, F2, F3, and F4 together with the medium M. On the other hand, the cell mass S larger than the outflow regions F1, F2, F3 and F4 is divided by the fibrous threads 27 and 28. Then, the cell masses S1, S2, S3, and S4 contained in the medium M flowing out from the opening region F have the same size as the outflow region F1, F2, F3, and F4 as seen from the direction D1, or the outflow region F1. , F2, F3, smaller than F4. Therefore, the size of the cell mass S can be contained within a desired range.

By the way, if the cell mass after division is too small, the cell mass may not survive. That is, when dividing the cell mass, the cell mass is divided by the fiber thread, but the cells in the portion in contact with the fiber thread are damaged. Then, when the area of the outflow region is small, the ratio of the portion in contact with the fiber thread to the cell mass after division increases, so that the cell mass is easily damaged. Therefore, when there is a magnitude relationship in the area of the outflow region, an outflow region in which the cell mass is easily damaged is generated.

On the other hand, in the split nozzle 23 according to the present embodiment, the areas of the outflow regions F1, F2, F3, and F4 are substantially equal to each other. Then, the sizes of the cell masses S1, S2, S3, and S4 after division become constant with each other. As a result, since there is no outflow region in which the cell masses S1, S2, S3, and S4 are easily damaged, the occurrence of cell death due to division can be reduced.

Further, one cell mass S comes into contact with two fiber threads 27 and 28. According to this configuration, the resistance force that the cell mass S receives from the fiber threads 27 and 28 can be reduced. Therefore, the pressure required for the cell mass S to pass through the opening region F is reduced. As a result, the damage to the cell mass S due to the pressure is reduced, so that the generation of dead cells can be further reduced.

Although the present disclosure has been described above, the present disclosure may be carried out in various forms without being limited to the configuration of the present disclosure.

<Modifications 1 and 2>
In the above embodiment, the cell division device 5 forms four outflow regions F1, F2, F3, and F4 in the opening region F. The number of outflow regions F1, F2, F3, and F4 is not limited to four as long as they have substantially the same area. For example, as shown in part (a) of FIG. 4, the dividing nozzle 23A of the cell dividing device 5A according to the first modification is provided with a dividing portion 26A having one fiber thread 27. According to this cell division device 5A, two outflow regions F5 and F6 are formed in the opening region FA, and the outflow regions F5 and F6 have an interval (angle KA) of 180 degrees around the axis A23. Therefore, the cell mass S can be divided into two.

Further, as shown in part (b) of FIG. 4, the dividing nozzle 23B of the cell dividing device 5B according to the second modification is provided with a dividing portion 26B having three fiber threads 33, 34, 35. These fiber threads 33, 34, and 35 extend radially from the axis A23 of the split nozzle 23B (the center of the opening region FB). Specifically, the outflow regions F7, F8, and F9 have an interval (angle KB) of 120 degrees around the axis A23. According to this cell division device 5B, three outflow regions F7, F8, and F9 are formed in the opening region FB. Therefore, the cell mass S can be divided into three parts.

Further, the opening region F through which the medium M flows out may be provided on the tube wall of the split nozzle 23.

1 Cell culture device 2 Cell culture tank 3 Air exposure tank 4 Waste liquid tank 5, 5A, 5B Cell division device 6 Medium outflow pipe 7 Medium introduction pipe 8 Medium discharge pipe 9 Lid 11 Pump 12 Gas supply pipe 13 Medium supply pipe 14 Air filter 16 Medium supply pipe 17 Pump 18 Medium discharge pipe 19 Medium recovery pipe 21 Front storage 22 Rear storage 23, 23A, 23B Divided nozzle (tube)
23a Tip portion 23b Outer peripheral surface 23c End surface 24 Flow path 26, 26A, 26B Divided portion 27 Fiber yarn (divided fiber portion, first fiber yarn)
28 Fiber yarn (split fiber part, second fiber yarn)
29 Thread arrangement part (fiber arrangement part)
31 Bands 33, 34, 35 Fiber yarn (split fiber part)
A23 Axis D1 Direction F, FA, FB Aperture area (opening)
F1, F2, F3, F4, F5, F6, F6, F8, F9 Outflow region K, KA, KB Angle M Medium R1, R2 Radial S, Sa, Sb, S1, S2, S3, S4 Cell clumps

Claims (6)

A cell division device that receives a medium containing a cell mass cultured in a cell culture tank and divides the cell mass so as to be smaller than a predetermined size.
A cylindrical tube portion having an opening for forming a flow path through which the medium flows and allowing the medium to flow out from the flow path, and a tubular portion.
A division portion provided in the opening and for dividing the cell mass flowing out from the opening together with the medium is provided.
The split fiber portion is formed around the axis of the pipe portion and is arranged across the opening so that at least two outflow regions having equal areas are formed in the opening. Including a cell division device. The cell division device according to claim 1, wherein the opening is provided at the tip of the tube. The split fiber portion has a first fiber thread and a second fiber thread arranged in the opening so that the four outflow regions are formed in the opening.
The cell division device according to claim 1 or 2, wherein the first fiber thread intersects the second fiber thread. The cell division device according to claim 3, wherein the position where the first fiber thread intersects with the second fiber thread is on the axis of the tube portion. The tube portion has a groove-shaped fiber arrangement portion provided on an end surface surrounding the opening.
The cell dividing device according to any one of claims 1 to 4, wherein the dividing fiber portion is arranged in the fiber arranging portion. A cell culture tank for culturing cell masses contained in the medium,
It is provided with a cell division device which is connected to the cell culture tank, receives the medium from the cell culture tank, and divides the cell mass into smaller than a predetermined size.
The cell division device
A cylindrical tube portion having an opening for forming a flow path through which the medium flows and allowing the medium to flow out from the flow path, and a tubular portion.
A division portion provided in the opening and for dividing the cell mass flowing out from the opening together with the medium is provided.
The split fiber portion is formed around the axis of the pipe portion and is arranged across the opening so that at least two outflow regions having equal areas are formed in the opening. Including a cell culture device.

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