Dispensing method and dispensing device

Abstract

Provided is a conduit system comprising: a dispensation liquid container having a liquid extraction port and a liquid return port; a circulation circuit extending from the liquid extraction port to the liquid return port; a plurality of branch circuits that are individually branched from the circulation circuit via respective circuit switching units; and dispensation containers that are connected to the leading ends of the respective branch circuits. A circulation step in which a cell solution in the dispensation liquid container flows in the circulation circuit, and, subsequently, a dispensing step in which the cell solution flows through dispensing flow paths to the respective dispensation containers are executed.

Description

Dispensing method and dispensing device

[0001] The present disclosure relates to a method for dispensing a predetermined amount of a mixture of a cell suspension and a cryopreservation solution into a plurality of dispensing containers to fill them evenly in the field of regenerative medicine, and to a dispensing device for carrying out this method.

[0002] In the medical field, it is common to divide a large amount of liquid (such as a pharmaceutical product) into portions that can be used in one go, and then seal and fill individual containers such as ampoules or vials for storage. To efficiently perform this process, a manifold with one inlet and multiple outlets is used, a container containing a large amount of liquid is connected to the inlet, and individual containers are connected to each of the multiple outlets, and the liquid is dispensed into the individual containers while switching the flow path by operating a valve. For example, Patent Document 1 discloses a system for aseptically filling vials with pharmaceutical products using this method.

[0003] Meanwhile, in the field of regenerative medicine, a mixture of a suspension of expanded cells and a cryopreservation solution (hereinafter referred to as "cell solution") is frozen and stored, and then administered to patients in need of regenerative medicine when needed. Conventionally, cell solution containing cells such as large-scale cultured MSCs (mesenchymal stem cells) is cryopreserved by manually dispensing (dividing) the solution into multiple dispensing containers while maintaining the cells in a suspended state.

[0004] Special Publication No. 2013-544139

[0005] In Patent Document 1, a container containing a large amount of pharmaceutical is connected to the inlet of a manifold having one inlet and multiple outlets (dispensing ports), small containers (vials) are connected to each of the multiple outlets, and the pharmaceutical is dispensed into the vials while switching the flow path by operating a valve. However, the pharmaceutical dispensed in Patent Document 1 is a solution, not a suspension, and does not contain living organisms such as living cells.

[0006] In contrast, cell suspensions are prepared by adding a cryopreservation solution to a cell suspension and mixing it. Because the cryopreservation solution is toxic to live cells, the cells must be dispensed into individual containers quickly (usually within one hour of preparation) and frozen for rapid storage. This dispensing process requires significant manpower, and operators must be extremely careful to ensure a consistent cell volume in each dispensing container to ensure a consistent supply of cells to patients. Furthermore, if cells suspended in the cell suspension settle and become densely packed, this can inhibit the respiration of live cells and cause damage. Furthermore, if the cell suspension density (hereinafter referred to as "cell concentration") becomes uneven, the amount dispensed into each dispensing container can become inconsistent. Furthermore, vigorous stirring to prevent cell settling in the cell suspension or sudden or excessive changes in the flow rate of the cell suspension must be avoided, as this can damage the live cells in the cell suspension.

[0007] Therefore, the technology disclosed herein aims to solve the above-mentioned problem of evenly dispensing a suspension of living cells, a living organism, into small containers such as vials or ampoules in a short amount of time without requiring the manual techniques of a large number of skilled workers.

[0008] The present disclosure provides a flow path system including a circulation circuit comprising a piping line connected to a liquid receiving container having a liquid withdrawal port and a liquid return port containing a cell liquid, the piping line extending from the liquid withdrawal port to the liquid return port, a liquid delivery pump provided midway through the circulation circuit, and a plurality of branch circuits each comprising piping lines branching out via a circuit switching unit connected to the circulation circuit between the outlet of the liquid delivery pump and the liquid return port, with a dispensing container connected to the tip of each branch circuit, and a dispensing container being connected to the end of each branch circuit, and a flow path system configured such that, by operating the circuit switching unit, a dispensing flow path from the liquid receiving container to each dispensing container and a circulation flow path returning to the liquid receiving container can be individually formed; The present invention provides a dispensing method comprising: an initial circulation step in which the circuit switching unit is operated and the liquid delivery pump is driven while stirring the liquid in the receiving liquid container to create a circulation state in which the cell liquid in the receiving liquid container is drawn out, flows through the circulation circuit, and is returned to the receiving liquid container; an initial dispensing step, following the initial circulation step, in which the circuit switching unit is operated to create a state in which the cell liquid is delivered to a dispensing container connected to the tip of one of the plurality of branch circuits; and at least one subsequent dispensing step, in which the circuit switching unit is operated after the initial dispensing step is completed to create a state in which the cell liquid is delivered to a dispensing container connected to the tip of another of the plurality of branch circuits, thereby solving the above-mentioned problem.

[0009] The present disclosure also provides a dispensing device for carrying out the above-mentioned dispensing method, which includes a circulation circuit consisting of a pipe extending from an inlet end to an outlet end, a liquid delivery pump provided midway through the circulation circuit, and a plurality of branch circuits consisting of pipes connected to the circulation circuit between the outlet of the liquid delivery pump and the outlet end via a circuit switching unit, wherein the liquid withdrawal port and liquid return port of a liquid container to be dispensed are connected to the inlet end and outlet end, respectively, and when a dispensing container is connected to each end of the plurality of branch circuits, a flow path system configured so that, by operating the circuit switching unit, a dispensing flow path from the liquid container to each dispensing container and a circulation flow path returning to the liquid container can be individually and selectively formed, and a liquid stirring means for shaking or pressing the liquid container to be dispensed connected to the circulation circuit, thereby solving the above-mentioned problem.

[0010] In fields such as regenerative medicine, when dividing a large amount of cell liquid prepared in a receiving liquid container such as a source bag into smaller portions and dispensing them into dispensing containers (individual containers) such as vials, the liquid can be dispensed evenly into multiple dispensing containers in a short amount of time without requiring many skilled workers.

[0011] 11 shows a basic form for carrying out the method of the present disclosure. 12 shows another form for carrying out the method of the present disclosure. 13 shows an operational flow in the basic form for carrying out the method of the present disclosure. 14 shows a timing chart of the basic form for carrying out the method of the present disclosure. 15 shows an example of a cell suspension state in a liquid transfer pipeline when carrying out the method of the present disclosure. 16 shows another example of a cell suspension state in a liquid transfer pipeline when carrying out the method of the present disclosure. 17 shows an operational flow in another form for carrying out the method of the present disclosure. 18 shows a timing chart of another form for carrying out the method of the present disclosure. 19 shows an example of a cell suspension state in a liquid transfer pipeline in another form for carrying out the method of the present disclosure. 20 shows the appearance of an example of a dispensing device for carrying out the method of the present disclosure. 21 shows the appearance of the cell liquid circulation circuit section of the dispensing device of FIG. 22. 22 shows the appearance of the cell liquid mixing section of the dispensing device of FIG. 23. 23 shows an example of a source bag connected to the dispensing device of FIG. 24. 24 shows an example of a dispensing container connected to the dispensing device of FIG. 25. 25 schematically shows the cell liquid circulation circuit and cell liquid mixing section of the dispensing device of FIG. 26. 26 shows the operational flow of the cell liquid circulation circuit of FIG. 27. 27 shows the operational flow of the cell liquid mixing section of FIG. 28. 9 shows the appearance of the ultrasonic sealer unit of the dispensing device of Figure 8 (before sealing). 10 shows the appearance of the ultrasonic sealer unit of the dispensing device of Figure 8 (after sealing). 11 is an operation timing chart of the ultrasonic sealer unit of the dispensing device of Figure 8. 12 shows the appearance of the dispensing container decant unit of the dispensing device of Figure 8. 13 is an operation timing chart of the dispensing container decant unit of the dispensing device of Figure 8. 14 shows the operation of the dispensing container decant unit of the dispensing device of Figure 8. 15 shows the appearance of the dispensing container cooling unit of the dispensing device of Figure 8. 16 is an operation timing chart of the dispensing container cooling unit of the dispensing device of Figure 8.

[0012] The present disclosure provides a method for dispensing (dividing) a large amount of cell fluid contained in a receiving fluid container into multiple dispensing containers. The cell fluid is a suspension containing a cell suspension and a cryopreservation solution in a predetermined ratio. The cell suspension is a suspension of desired cells (e.g., cells expanded and cultured for administration to patients in regenerative medicine) in a liquid medium. The cryopreservation solution is a preservation liquid such as dimethyl sulfoxide (DMSO) that is added when freezing and preserving a cell suspension. In this specification, a receiving fluid container refers to a container such as a bag (hereinafter also referred to as a "source bag") that contains the cell fluid dispensed by the method of the present disclosure. Furthermore, a dispensing container refers to a small container such as a vial or an ampule that individually contains each of the cell fluid dispensed by the method of the present disclosure.

[0013] The disclosed method first includes a flow path preparation step of preparing a flow path system connecting a receiving liquid container containing a cell liquid to a plurality of dispensing containers. The receiving liquid container has a liquid withdrawal port and a liquid return port, and the flow path system has a circulation circuit consisting of a conduit connecting the liquid withdrawal port to the liquid return port. A liquid delivery pump is provided midway through the circulation circuit, and a plurality of branch circuits consisting of individually branched conduits are connected to the circulation circuit between the outlet of the liquid delivery pump and the liquid return port via a circuit switching unit (a flow path switching mechanism such as a three-way stopcock). One of the plurality of dispensing containers is connected to the tip of each branch circuit, and by operating the circuit switching unit, a dispensing flow path from the receiving liquid container to each dispensing container and a circulation flow path returning to the receiving liquid container can be individually formed.

[0014] The method of the present disclosure then includes an initial circulation step in which the circuit switching unit is operated and the liquid supply pump is driven to create a circulation state in which the cell fluid in the receiving liquid container is drawn out, flows through the circulation circuit, and is returned to the receiving liquid container. At this time, the cell fluid in the receiving liquid container is continuously or intermittently stirred to homogenize the cell concentration of the cell fluid in the receiving liquid container and the circulation circuit.

[0015] The method of the present disclosure then includes an initial dispensing step of operating the circuit switching unit and the liquid supply pump to supply the cell solution to a dispensing container connected to the tip of one of the branch circuits. This initial dispensing step begins when the cell concentration of the cell solution in the receiving container and the circulation circuit reaches a predetermined level in the initial circulation step.

[0016] The method of the present disclosure then includes a subsequent dispensing step of operating the circuit switching unit and the liquid supply pump to supply the cell solution to a dispensing container connected to the tip of another one of the branch circuits. This subsequent dispensing step may be performed immediately after the initial dispensing step, but is preferably performed after a circulation step similar to the initial circulation step, in order to more uniformly distribute the cell concentration of the cell solution in the dispensing container and the circulation circuit.

[0017] In the method disclosed herein, dispensing into two dispensing containers is completed after the above-mentioned subsequent dispensing step has been performed once, but by then repeating the above-mentioned subsequent dispensing step as many times as necessary, dispensing into the desired number of dispensing containers can be performed.

[0018] Specific embodiments for implementing the method of the present disclosure are described below. FIG. 1A is a schematic diagram illustrating a basic embodiment for implementing the method of the present disclosure. A cell solution 117, a mixture of a cell suspension and a cryopreservation solution, is contained in a source bag 118, which is a liquid receiving container having a liquid withdrawal port and a liquid return port. The cell solution 117 is stirred by a cell solution stirring unit 116 so that the cell concentration is uniform within the source bag 118. The cell solution 117 withdrawn from the liquid withdrawal port of the source bag 118 is individually delivered to multiple dispensing containers (vials) 131 by a liquid delivery pump 111a. Note that while five vials are shown in FIG. 1A, the number of dispensing containers is not limited to five and may be 11 (one of which is for quality control), as described below.

[0019] The dispensing container 131 to which the cell fluid 117 is to be sent is selected by switching the flow path using a circuit switching unit (a three-way stopcock in the figure) 115c provided at each branch point to the multiple dispensing containers 131. Alternatively, the cell fluid 117 sent from the source bag 118 can be returned to the source bag 118 by going around the circulation circuit 10 without branching at the branch points to each dispensing container 131.

[0020] The source bag 118 is equipped with a source bag cooling unit 119 that maintains the source bag 118 at a constant temperature (e.g., 2°C to 8°C). The circulation circuit 10, which is the conduit through which the cell fluid 117 drawn from the fluid drawing port of the source bag 118 flows, is equipped with a flow rate measuring unit 112 that measures the flow rate of the cell fluid 117 in real time and, based on that, accurately monitors the amount of cell fluid that has entered each dispensing container 131. The cell fluid 117 remaining in the circulation circuit 10 after being delivered to each dispensing container 131 is returned to the source bag 118 via the circulation circuit 10 and stirred by the cell fluid stirring unit 116 so that the cell concentration in the source bag 118 becomes uniform.

[0021] 1B is a schematic diagram illustrating a configuration in which a conduit (another circulation circuit 113) for circulating the cell solution in the dispensing container via a route separate from the circulation circuit 10 is added to the configuration of FIG. 1A. In this configuration, the cell solution 117 in the source bag 118 can be drawn from the bottom of the source bag 118 and returned to the top of the source bag 118 via the other circulation circuit 113 having the liquid supply pump 111b. The basic operation of the device in FIG. 1B is the same as that of the device in FIG. 1A, but by optionally circulating only the inside of the source bag 118, the concentration of the cell solution 117 in the source bag 118 can be made more uniform.

[0022] FIG. 2 shows a flow (procedure) for dispensing (transporting) the cell fluid 117 from the source bag 118 to each dispensing container 131 using the device of FIG. 1A or 1B. Here, it is assumed that 11 dispensing containers 131 are connected to the circulation circuit 10 via the circuit switching unit 115c. However, the same applies when five dispensing containers are connected, as shown in FIG. 1A or 1B. First, the source bag 118 containing the cell fluid 117 is placed in a cell fluid mixing unit capable of stirring and cooling (temperature control) the cell fluid. Then, the device waits until the temperature of the source bag 118 reaches a predetermined temperature (2°C to 8°C) while stirring the cell fluid. If the set temperature has been reached but the cell fluid stirring has not yet reached the predetermined operating time, stirring continues until the predetermined operating time is reached. Next, the fluid feed pump 111a is rotated at high speed, circulating the cell fluid 117 through the circulation circuit 10 for a predetermined time. Thereafter, the liquid feed pump 111a is switched to low speed rotation, and the dispensing operation to each dispensing container 131 begins. First, the circuit switching unit (three-way stopcock) 115c at dispensing position #1 is switched from a closed state (circulation side) to an open state (dispensing side), and liquid feed to the dispensing container 131 connected to the end of the branch circuit branching at dispensing position #1 begins. While liquid feed is being performed, the flow rate measuring unit 112 measures the flow rate of the cell liquid 117 in real time, and the amount of liquid fed to the dispensing container is constantly monitored based on the integrated flow rate calculated from the measurement. When the amount of liquid fed to the dispensing container 131 at dispensing position #1 reaches a predetermined amount, the circuit switching valve 115c at dispensing position #1 is switched to a closed state (circulation side), and dispensing to the dispensing container is completed. The same operations as those performed for dispensing position #1 are repeated sequentially for dispensing positions #2 to #11, and then the liquid feed pump 111a is stopped.

[0023] If the amount of cell liquid 117 dispensed (liquid delivery amount) into each dispensing container does not reach the specified amount or exceeds the specified amount, the liquid delivery pump 111a is immediately stopped to stop dispensing.

[0024] Figure 3 shows the detailed operational timing of the above procedure. Here, the connecting positions of the 11 vials are numbered #1 to #10 and QC, and the QC vial is not shown. The cell solution 117 is circulated for a predetermined time (approximately 30 seconds) at high speed (35 ml / min) by the liquid pump 111a. After that, the circuit switching valve (three-way stopcock) 115b of the circulation circuit 10 is switched OFF (circulation side open → closed), and then the circuit switching valve 115c at dispensing position #1 is switched ON (dispensing side closed → open), and the liquid pump 111a is switched to a low speed (15 ml / min) to deliver the solution to the dispensing container 131 at dispensing position #1. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #1 is switched OFF (dispensing side open → closed), the circuit switching valve 115c at dispensing position #2 is switched ON (dispensing side closed → open), the liquid transfer pump 111a is set to low speed (15 ml / min) to transfer the liquid to the dispensing container 131 at dispensing position #2. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #2 is switched OFF (dispensing side open → closed), the circuit switching valve 115c at dispensing position #3 is switched ON (dispensing side closed → open), the liquid transfer pump 111a is set to low speed (15 ml / min) to transfer the liquid to the dispensing container 131 at dispensing position #3. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #3 is switched OFF (dispensing side open → closed). After that, the same operation is repeated from dispensing position #4 to #11 (QC). After the liquid has been sent to all the dispensing containers, the liquid sending pump 111a is switched from low speed (15 ml / min) to high speed (35 ml / min), and the remaining cell liquid 117 is circulated for a predetermined time (about 30 seconds in this case).

[0025] FIG. 4A shows how the suspension state of cells 11 in the cell fluid 12 (cell fluid 117) in the fluid feed line 15 (circulation circuit 10) changes over time during the fluid feed operation. If the fluid is fed when the cell concentration of the cell fluid 117 in the source bag 118 is non-uniform, the cell concentration of the cell fluid 12 varies depending on the position in the fluid feed line 15, resulting in a non-uniform dispersion state, as shown in step 1 of FIG. 4A . Therefore, to reduce the variation in the cell concentration of the cell fluid 12 in the fluid feed line 15, the fluid feed pump is rotated at high speed to circulate the cell fluid 117 within the circulation circuit 10, and the cell concentration of the cell fluid 12 in the fluid feed line 15 is homogenized, as shown in step 2. Thereafter, when the cell concentration of the cell fluid 12 in the fluid feed line 15 is homogenized, the fluid feed pump is switched from high speed to low speed, and the circuit switching valve 13 (111c) is set to the dispensing side, as shown in step 3, to feed the fluid to the dispensing container 14 (131). Then, when dispensing is performed successively into a plurality of dispensing containers 14, as shown in step 4, the variation in cell concentration among the plurality of dispensing containers 14 after dispensing is reduced.

[0026] 4B shows the state of the cell fluid 12 in the fluid feed conduit 15 when the fluid feed conduit 15 has a curved portion. In this case, too, the cell concentration of the cell fluid 12 in the fluid feed conduit 15 before dispensing varies depending on the position within the fluid feed conduit 15, with the cell concentration being particularly high at the curved portion of the fluid feed conduit 15. If the cell fluid 12 is fed to multiple dispensing containers 14 under such conditions, the variation in cell concentration between the dispensing containers 14 will increase. That is, at the beginning of the fluid feed, the cell concentration of the cell fluid 12 in the fluid feed conduit 15 will be non-uniform depending on the position within the fluid feed conduit 15, as shown in step 1. Therefore, to reduce the variation in the cell concentration of the cell fluid 12 in the fluid feed conduit 15, the fluid feed pump is rotated at high speed to circulate the cell fluid 117 within the circulation circuit 10, thereby making the cell concentration uniform within the fluid feed conduit 15, including the curved portion, as shown in step 2. Thereafter, as shown in step 3, the liquid delivery pump is switched from high speed to low speed, and the circuit switching valve 13 is set to the dispensing side to deliver liquid to the dispensing containers 14. Then, as shown in step 4, when dispensing is performed continuously to multiple dispensing containers 14, the variation in cell concentration among the multiple dispensing containers 14 after dispensing is reduced.

[0027] FIG. 5 illustrates another flow (procedure) for dispensing (transferring) the cell fluid 117 from the source bag 118 to the dispensing container 131. Here, eleven dispensing containers 131 (dispensing positions #1 to #10 and a QC vial) are connected, and dispensing (transferring) is performed sequentially from the vial at dispensing position #10 to the vial at dispensing position #1 (in the opposite direction to that shown in FIG. 2), and finally to the QC vial. First, the source bag 118 containing the cell fluid 117 is placed in a cell fluid mixing section capable of stirring and temperature control (cooling) the cell fluid. Then, while stirring the cell fluid 117, the system waits until the temperature of the source bag 118 reaches a predetermined temperature (2°C to 8°C). Furthermore, if the set temperature has been reached but the cell fluid stirring operation time has not yet reached the predetermined operating time, the stirring operation continues until the predetermined operating time is reached. Next, the liquid feed pump 111a is rotated at high speed, and the cell fluid 117 is circulated through the circulation circuit 10 for a predetermined period of time. The liquid delivery pump 111a is then switched to low speed rotation, and the dispensing operation begins. Specifically, the circuit switching valve (three-way stopcock) 115c at dispensing position #10 is switched ON (dispensing side closed → open) to begin delivering liquid to the dispensing container. While delivering liquid, the flow rate measurement unit 112 measures the flow rate of the cell liquid 117 in real time, and the amount of delivered liquid is constantly monitored based on the integrated flow rate calculated from the flow rate. When the amount of delivered liquid to the dispensing container 131 at dispensing position #10 reaches a predetermined amount, the circuit switching unit 115c at dispensing position #10 is switched OFF (dispensing side open → closed), and dispensing to the dispensing container is terminated. The above operations performed for dispensing position #10 are repeated from dispensing position #9 to dispensing position #1, and finally, dispensing is performed to the dispensing container 131 at dispensing position 11 (for QC), after which the liquid delivery pump 111a is stopped. If the amount of cell liquid 117 dispensed (liquid delivery amount) into each dispensing container does not reach a predetermined amount or exceeds the predetermined amount, the liquid delivery pump 111a is stopped immediately.

[0028] Figure 6 shows the detailed operational timing of the above-described procedure. Here, the positions connecting the 11 vials are numbered #1 to #11 (#11 is for QC). The cell solution 117 is circulated for a predetermined time (approximately 30 seconds) at high speed (35 ml / min) by the liquid delivery pump 111a. After that, the circuit switching valve (three-way stopcock) 115b of the circulation circuit 10 is switched OFF (circulation side open → closed), and then the circuit switching valve 115c at dispensing position #10 is switched ON (dispensing side closed → open), and the liquid delivery pump 111a is switched to a low speed (15 ml / min) to deliver the liquid to the vial at dispensing position #10. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #10 is switched OFF (dispensing side open → closed), the circuit switching valve 115c at dispensing position #9 is switched ON (dispensing side closed → open), and the liquid transfer pump 111a is rotated at a low speed (15 ml / min) to transfer the liquid to the vial at dispensing position #9. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #9 is switched OFF (dispensing side open → closed), and the circuit switching valve 115c at dispensing position #8 is switched ON (dispensing side closed → open), and the liquid transfer pump 111a is rotated at a low speed (15 ml / min) to transfer the liquid to the vial at dispensing position #8. When the liquid transfer is complete, the circuit switching valve 115c at dispensing position #8 is switched OFF (dispensing side open → closed). Thereafter, the same operation is repeated (in descending order) up to dispensing position #1. Finally, after dispensing at dispensing position #11 (for QC) is completed, the liquid feed pump 111a is switched from low speed (15 ml / min) to high speed (35 ml / min), and the remaining cell liquid 117 is circulated for a predetermined time (here, about 30 seconds).

[0029] 7 shows the state of cell suspension in the liquid feed conduit 15 when the operations of Figures 5 and 6 are performed. When the cell concentration of the cell solution 117 in the source bag 118 is non-uniform and dispensing is performed to multiple (here, 11) dispensing containers 14 (131) in order from the dispensing container with the longest liquid feed path from the source bag (i.e., in descending order from the dispensing container 14 at dispensing position #10 to the dispensing container 14 at dispensing position #1), when dispensing to the previous dispensing container is completed and dispensing is switched to the next dispensing container, the cell solution 12 in the liquid feed conduit 15 must be returned. This results in a non-uniform dispersion state of cells in the cell solution coming out of the source bag, with the dispersion density varying depending on the position in the liquid feed conduit 15. Therefore, in order to reduce the variation in cell concentration of the cell fluid 12 in the fluid feed line 15, the fluid feed pump is rotated at high speed to circulate the cell fluid 117 within the circulation circuit 10, and the cell concentration of the cell fluid 12 in the fluid feed line 15 is made uniform as shown in step 2. Then, once the cell concentration of the cell fluid 12 in the fluid feed line 15 has become uniform, the fluid feed pump is switched from high speed to low speed as shown in step 3, and the circuit switching valve 13 is set to the dispensing side to feed the fluid to the dispensing container 14. Then, when dispensing is performed continuously into multiple dispensing containers 14, the variation in cell concentration among the multiple dispensing containers 14 after dispensing is reduced as shown in step 4.

[0030] Cell solution was dispensed from a source bag into multiple dispensing containers (five vials) of the same shape and volume, twice each, using the conventional method without circulating the cell solution and the method of the present disclosure with circulating the cell solution. The results of measuring the number of cells in each dispensing container for each case are shown below. Table 1 shows the results of dispensing using the conventional method, and Table 2 shows the results of dispensing using the method of the present disclosure.

[0031]

[0032]

[0033] In Tables 1 and 2, "vial No." refers to the number indicating the branch position of the branch circuit to which the vial is connected. Vial No. 1 is connected to the end of the branch circuit that branches at a position closest to the source bag, and vial No. 5 is connected to the end of the branch circuit that branches at a position farthest from the source bag. "Mean value" refers to the arithmetic mean of the cell counts from the first and second runs (the overall mean is the arithmetic mean of the cell counts for all 10 runs), "deviation" refers to the difference from the mean (the overall deviation is the arithmetic mean of the differences between the cell counts for all 10 runs and the overall mean, i.e., the mean deviation; the deviations for the first and second runs for the same vial No. are equal), and "CV value" refers to the coefficient of variation (deviation / mean value).

[0034] Essentially, the dispensing accuracy of the cell count in each dispensing container during dispensing, i.e., the coefficient of variation (CV), should be within 10%. While the overall CV for the conventional technology shown in Table 1 is 12%, which does not reach the predetermined target value, the overall CV for the technology of the present disclosure shown in Table 2 is 8%, which achieves the predetermined target value. Incidentally, the arithmetic mean (corresponding to intraclass variation) of the CV values ​​for vials No. 1 to No. 5 is 4.4% for the conventional technology shown in Table 1 and 5.0% for the technology of the present disclosure shown in Table 2, indicating that there is essentially no difference between the conventional technology and the technology of the present disclosure. Therefore, the large difference (approximately 1.5 times) in the overall CV between the conventional technology and the technology of the present disclosure clearly demonstrates that the technology of the present disclosure reduces variations in cell count due to differences in vial position compared to the conventional technology.

[0035] Next, a dispensing device for carrying out the method of the present disclosure will be described. The method of the present disclosure includes a flow path preparation step in which a flow path system having a predetermined configuration including a circulation circuit is prepared, and an initial circulation step, an initial dispensing step, and a subsequent dispensing step in which the flow path system is operated in a predetermined sequence. Therefore, the dispensing device for carrying out the method of the present disclosure can be said to include the flow path system prepared in the flow path preparation step. However, since the liquid receiving container and the multiple dispensing containers are connected or linked to the flow path system when carrying out the method of the present disclosure, they are not components of the dispensing device. However, since at least the initial circulation step of the method of the present disclosure requires continuous or intermittent stirring of the cell fluid in the liquid receiving container, a necessary component of the device for carrying out the method of the present disclosure is a mechanism for continuously or intermittently stirring the cell fluid in the liquid receiving container when the liquid receiving container is connected to the flow path system.

[0036] Specifically, a dispensing device for carrying out the method of the present disclosure includes a flow path system having a predetermined configuration including a circulation circuit and a liquid agitation means for agitating the cell liquid in the receiving liquid container connected to the circulation circuit. The flow path system includes a circulation circuit consisting of a conduit extending from an inlet end to an outlet end, a liquid delivery pump provided along the circulation circuit, and multiple branch circuits consisting of conduits connected to the circulation circuit between the outlet of the liquid delivery pump and the outlet end via a circuit switching unit. The flow path system is configured such that the liquid withdrawal port and liquid return port of the receiving liquid container are connected to the inlet end and outlet end of the circulation circuit, respectively, and when a dispensing container is connected to each end of the multiple branch circuits, the circuit switching unit can be operated to individually and selectively form a dispensing flow path from the receiving liquid container to each dispensing container and a circulation flow path returning to the receiving liquid container. The liquid agitation means is configured to agitate the cell liquid in the receiving liquid container by shaking or pressing the receiving container connected to the circulation circuit.

[0037] An important indicator for determining whether a dispensing device for carrying out the method of the present disclosure is suitable is whether it can quickly dispense a large amount of cell fluid, which is a mixture of a cell suspension and a cryopreservation solution in a ratio of 9:1, while maintaining cell quality. While there is an indicator for the accuracy of dispensing the volume of cell fluid that a CV value of 5% or less is used, the indicator for the accuracy of dispensing the number of cells in the cell fluid remains unclear at present.

[0038] Figure 8 shows an example of the appearance of a dispensing device suitable for carrying out the method of the present disclosure. The dispensing device 100 shown in Figure 8 has a main plate (vertical base) 100b fixed to the upper surface of a main frame (support base) 100a. A control system controller and auxiliary units are also attached to the main frame 100a. Meanwhile, functional units such as a cell fluid circulation circuit unit 110, a dispensing container sealer unit 130, a dispensing container decant (shaking) unit 140, a dispensing container cooling unit 150, and a cell fluid mixing and stirring unit 160 are attached to the main plate 100b, and each functional unit can be retrofitted.

[0039] 9A shows the external appearance of the cell fluid circulation circuit unit 110. The cell fluid circulation circuit unit 110 constitutes a flow path system that transfers cell fluid 117, a mixture of a cell suspension and a cryopreservation solution, from a source bag 118 to multiple dispensing containers 131. The cell fluid circulation circuit unit 110 is composed of a circuit piping unit 113 (circulation circuit 10 and branch circuit), a circuit holder unit 114 that holds the circuit piping unit in a predetermined position (basically horizontal), a fluid transfer pump 111 that transfers the cell fluid 117 from the source bag 118, a flow rate measuring unit 112 that instantaneously measures the flow rate of the transferred cell fluid 117 and calculates the transfer amount as an integrated flow rate, and a circuit switching unit 115 that switches the flow path to dispense the cell fluid 117 flowing in the separate circulation circuit 113 into multiple dispensing containers 131.

[0040] FIG. 9B shows the detailed configuration of the cell fluid mixing / agitating unit 160. The source bag 118 is fixed to the vertically driven support base 166 by the source bag gripper 164. The fixed source bag 118 is cooled and maintained at a predetermined temperature (e.g., 2°C to 8°C) by the source bag cooling unit 162. The source bag cooling unit 162 cools the source bag 118 using a Peltier element structure that uses water or air cooling. Furthermore, in regenerative medicine, condensation is undesirable for bacterial growth. Therefore, even if condensation occurs on the source bag gripper 164 when the delivery of the cell fluid 117 inside the source bag 118 has been completed, a reverse current is passed through the Peltier element to raise the temperature, evaporating the water and suppressing bacterial growth.

[0041] The sauce bag temperature detector 161 constantly monitors the surface temperature of the sauce bag 118 cooled by the sauce bag cooler 162, and performs feedback control to maintain a predetermined temperature. If the temperature is not within the predetermined temperature range (for example, 2°C to 8°C), an abnormality signal is sent.

[0042] When the surface temperature of the source bag 118 reaches a predetermined temperature range, the source bag swing drive unit 163 tilts the source bag 118 to one side (e.g., left) at a predetermined angle (usually within 50°) and maintains this tilted state for a certain period of time (30 seconds to 2 minutes). Next, the source bag swing drive unit 163 tilts the source bag 118 to the other side (e.g., right) at a predetermined angle (usually within 50°) and maintains this tilted state for a certain period of time (30 seconds to 2 minutes). This swinging motion from side to side is repeated several times, and when the cell concentration of the cell fluid 117 becomes constant, the source bag open / close valve is opened and the delivery of the cell fluid 117 from the source bag 118 begins. Thereafter, the source bag swing drive unit 163 continues to perform the swinging motion, thereby continuing the delivery of the cell fluid to the cell fluid circulation circuit unit 110. When the amount of cell liquid 117 in the source bag 118 decreases, the source bag vertical drive unit 165 tilts the source bag 118 from the horizontal position diagonally so that the liquid outlet is downwards. This source bag tilting operation is performed using the link mechanism of the source bag vertical drive support base 166.

[0043] During the supply of the cell fluid to the cell fluid circulation circuit section 110, the source bag flow rate detection section 167 constantly monitors the flow rate of the cell fluid 117 sent out from the source bag 118 to ensure that it remains constant, and if the flow rate fluctuates, it adjusts it by sending feedback to the source bag up / down drive section 165 so that the flow rate of the cell fluid 117 remains constant. Also, since the volume of the cell fluid 117 in the source bag 118 is clear in the initial state, the source bag flow rate detection section 167 manages the volume of the cell fluid 117 sent from the source bag 118 using the following formula: Formula: Initial volume of cell fluid in source bag = volume of cell fluid sent + volume of cell fluid in source bag

[0044] FIG. 10A shows details of a source bag 118 containing cell fluid 117. The source bag 118 is composed of a source bag body 20, an upper insertion tube 21, and a lower discharge tube 22. The outer periphery of the source bag body 20 is glued together to form a source bag outer periphery 23, which is fixed to the cell fluid mixing and stirring unit 160 by clamping it with a source bag gripper 164. Because cells (especially adherent cells such as MSCs) tend to adhere to the inner surface of the source bag body 20, the inner surface of the source bag body 20 is surface-treated with a coating agent 24 (e.g., POC coating or fluorine coating) to prevent cell adhesion. The area near the outlet of the source bag body 20, to which the lower discharge tube 22 is attached, has an outlet angle of 45° to 60° to facilitate the discharge of cells in the cell fluid, and is shaped to eliminate flat surfaces to prevent cell fluid 117 from remaining.

[0045] In addition, the length of the upper insertion tube 21 is set to extend into the source bag body 20 by a predetermined length (approximately 80% of the bag height) so that the cells do not easily adhere to the inner wall of the source bag body 20 when the amount of cell liquid in the source bag body 20 becomes low.

[0046] Furthermore, if the cell fluid 117 discharged from the lower discharge tube 22 and circulated back to the upper insertion tube 21 is allowed to pass through the tube cell fluid diffusion section 25 attached to the tip of the upper insertion tube 21 and strike the inner surface of the source bag body 20 at a constant flow rate, convection of the cell fluid 117 will occur within the source bag 118, facilitating uniform cell concentration. To achieve this, it is recommended to provide slits 26 with a width of approximately 0.5 to 1 mm in the tube cell fluid diffusion section 25, dividing the tube into four equal parts circumferentially. Providing such slits 26 not only uniforms the cell concentration through convection of the cell fluid 117, but also serves to prevent backflow of the cell fluid 117 when it becomes necessary to remove air from the source bag 118. Furthermore, if the outlet end of the source bag body 20 and the inlet end of the lower discharge tube 22 are smoothly connected with the same dimensions and shape, all of the cells in the cell fluid 117 can be discharged without leaving any residue. It is preferable that the source bag capacity be approximately 0.3 L to 50 L.

[0047] FIG. 10B illustrates various dispensing containers 131 for containing and storing a predetermined amount of dispensed cell fluid 117. In FIG. 10B, a vial 14 and a bag 41 are shown as representative dispensing containers 131, but the dispensing containers are not limited to these and may also be bottles or the like. Furthermore, the dispensing container 131 must be designed to be cryopreserved at temperatures between -80°C and -196°C. Therefore, the materials used for the dispensing container 131 are limited, typically glass or resin. In the case of resin, only glass fiber materials, EVA, or other materials that can withstand extremely low temperatures are used.

[0048] As shown in Figure 10B, representative dispensing containers, such as vials 14 and bags 41, are available with and without filters. In the filter-equipped vial shown in the upper left column of the figure, the delivered cell fluid 117 is injected through the individual container inlet tube 32a. In this case, air inside the vial 14 is vented to the outside through a filter 31 inserted into the individual container outlet tube 32b. In the filter-less vial shown in the upper right column of the figure, a bifurcated fitting 33 is attached to the inlet of the vial 14, one end of which is connected to the individual container inlet tube 32a and the other end to the individual container outlet tube 32b. The delivered cell fluid 117 is injected through the individual container inlet tube 32a, and air inside the vial 14 is vented to the outside through the individual container outlet tube 32b. In the filter-equipped bag shown in the lower left column of the figure, the delivered cell fluid 117 is injected through the individual container inlet tube 32a. In this case, the air inside the bag 41 is exhausted to the outside through the filter 31 inserted into the individual container OUT tube 32b. In the filter-less bag shown in the lower right column of the figure, a bifurcated joint 33 is attached to the inlet of the bag 41, one end of which is connected to the individual container IN tube 32a and the other end to the individual container OUT tube 32b. The delivered cell solution 117 is injected through the individual container IN tube 32a, and the air inside the bag 41 is exhausted to the outside through the individual container OUT tube 32b.

[0049] Next, one embodiment of an apparatus for carrying out the method of the present disclosure will be described with reference to Figures 11, 12A, and 12B. Figure 11 is a diagram schematically illustrating the cell solution circulation circuit unit 110 in the dispensing apparatus of this embodiment. Figure 12A is a diagram illustrating the operation flow of the cell solution circulation circuit unit 110 in the dispensing apparatus of this embodiment. Figure 12B is a diagram illustrating the operation flow of the source bag stirring in the dispensing apparatus of this embodiment.

[0050] First, the source bag stirring operation flow of the dispensing device of this embodiment, shown in FIG. 12B , will be described. A source bag 118 containing a cell solution 117, a mixture of cell suspension and cryopreservation solution at a ratio of 9:1, is placed on the vertical drive support base 166 of the cell solution mixing and stirring unit 160, lying approximately horizontally (at an angle of approximately 5° relative to the horizontal) with the lower discharge tube 22 slightly downward. This prevents cells in the cell solution 117 from remaining near the lower discharge tube 22 of the source bag 118 due to gravity. Furthermore, the source bag 118 must be cooled to suppress cell activation in order to reduce damage to the cells caused by the cryopreservation solution. To this end, a source bag cooling unit 119 (162) equipped with a Peltier element is brought into contact with the source bag 118 placed on the vertical drive support base 166, and the temperature of the Peltier element is set to a predetermined temperature (2°C to 8°C). The system then waits, maintaining this state, until the surface temperature of the source bag 118 (measured by the source bag temperature detector 161) reaches the predetermined temperature. Once the temperature measured by the source bag temperature detector 161 reaches the predetermined temperature, the cell fluid 117 in the source bag 118 is stirred in the cell fluid mixing and stirring unit 160 until the cell concentration of the cell fluid 117 in the source bag 118 becomes uniform. Stirring methods include a method of uniforming the cell concentration by periodically rocking the cell fluid 117 back and forth, a method of pressing the source bag 118, and a method of circulating the cell fluid 117 in the source bag 118 via a separate circulation circuit 113, as shown in FIG. 1B. After determining that the cell concentration of the cell fluid 117 has become uniform in the source bag stirring operation flow according to the above procedure, the system transitions to the operation flow of the cell fluid circulation circuit.

[0051] As shown in FIG. 12A , once the source bag 118 has cooled to a predetermined temperature and the cell concentration in the cell fluid 117 has become uniform, a leak check of the cell fluid circulation circuit unit 110 is then performed. The purpose of the leak check is to confirm whether there are any defects (such as voids) in the circuit of the cell fluid circulation circuit unit 110 itself before the cell fluid 117 in the source bag 118 is flowed through the cell fluid circulation circuit unit 110. The leak check procedure will be explained using FIG. 11 , which schematically illustrates the cell fluid circulation circuit unit 110 in the dispensing device of this embodiment. Note that while FIG. 11 depicts five dispensing containers connected together, the number of connected dispensing containers is not limited to five; for example, eleven dispensing containers may be connected together, as numbered No. 1 through No. 11 in FIG. 11 .

[0052] First, circuit switching unit 115a is switched to an open state, allowing air to be taken in from the atmosphere through filter 121a, rather than the inflow side from source bag 118. Then, all circuit switching units 115c are switched to an open state, allowing ventilation to vial 131. After that, circuit switching unit 115b is switched to an open state, opening to the atmosphere through filter 121b, rather than the circulation side to source bag 118. Furthermore, circuit switching unit 115d is switched to an open state, opening to the atmosphere through filter 121c, rather than the inflow side to source bag 118. By following the above procedure, residual pressure in the circuit is released.

[0053] Next, while circuit switching unit 115a remains open, circuit switching unit 115d switches filter 121c from open to closed, circuit switching unit 115b switches filter 121b from open to closed, and circuit switching unit 115c switches all of its circuits from open to closed (circulation side).

[0054] Next, while maintaining the circuit switching units 115a-115d in their current state, drive the fluid supply pump 111. This causes the value of the pressure sensor 122 to rise, and if the value indicated by the pressure sensor 122 reaches 250-300 kPa, the leak check is successful. If the value indicated by the pressure sensor 122 does not reach 250 kPa, there is a possibility of a defect (such as a leak) in the circulation circuit 10 of the intercellular fluid circulation circuit unit 110, and therefore use of this intercellular fluid circulation circuit unit 110 should be avoided.

[0055] If the leak check is successful, the circulation and dispensing of the cell solution begins. The circulation and dispensing procedures will be explained below with reference to Figures 11 and 12A.

[0056] First, the upper insertion tube 21 and lower discharge tube 22 extending from the source bag 118 are connected to the outlet and inlet sides of the circulation circuit 10, respectively, via sterile connectors 123. A sterile connection device can be used instead of the sterile connectors 123. Then, circuit switching unit 115a is switched from an open state, which takes in air through filter 121a, to a closed state, which switches the source bag 118 to the inlet side via circuit switching unit 115f, which serves as the outlet valve. Circuit switching unit 115b is also switched from an open state, which opens the source bag 118 to the atmosphere, via filter 121b, to a closed state, which switches the source bag 118 to the circulating side via circuit switching unit 115e, which serves as the inlet valve. Circuit switching unit 115d is also switched from an open state, which opens the source bag 118 to the atmosphere, via filter 121c, to a closed state, which switches the source bag 118 to the returning side via circuit switching unit 115e, which serves as the inlet valve.

[0057] Next, the source bag 118 is cooled and waited for to reach a predetermined set temperature (2° C. to 8° C.) Once the temperature of the source bag 118 reaches the predetermined set temperature, the cell liquid is stirred in the cell liquid mixing and stirring unit 160 until the cell concentration becomes constant.

[0058] Next, the liquid feed pump 111 is driven at high speed (70 to 100 mL / min) to circulate the cell liquid 117 within the cell liquid circulation circuit unit 110. The time for circulating the cell liquid 117 depends on the type of cells in the cell liquid 117, but is approximately 30 seconds to 3 minutes as a guide. This ensures that the cell concentration within the cell liquid circulation circuit unit 110 is equivalent between the source bag 118 and the circulation circuit 10.

[0059] Then, when the cell concentration in the source bag 118 and in the cell fluid circulation circuit section 110 including the circulation circuit 10 becomes roughly equal and uniform due to the circulation of the cell fluid 117 for a predetermined time, the liquid delivery pump 111 is switched to low-speed operation (15 to 70 mL / min) and dispensing into each vial begins.

[0060] Dispensing into each vial is performed, for example, in the following manner: (1) First, the circuit switching unit 115c at the No. 1 position is switched from a closed state to an open state, and the liquid feed pump 111 is driven at a low speed to feed the cell solution 117 into the dispensing container 131 at the No. 1 position. In this case, for vials with a small dispensing volume (e.g., 5 mL), the liquid feed rate of the liquid feed pump 111 is slowed (e.g., to 15 mL / min) to keep the cell concentration variation in the dispensing container 131 within 5%. On the other hand, for dispensing containers with a large dispensing volume, the drive speed of the liquid feed pump is increased (e.g., to 70 mL / min) to keep the cell concentration variation in the dispensing container 131 within 5%. (2) After dispensing of the cell solution 117 into the dispensing container 131 at the No. 1 position is completed, the dispensing container sealer unit 130 seals the tube of the dispensing container 131 at the No. 1 position. (3) The circuit switching unit 115c at the No. 1 position is switched from an open state to a closed state, and the circuit switching unit at the No. 2 position is switched from a closed state to an open state, and the liquid feed pump 111 is driven at a low speed to feed the cell solution 117 to the dispensing container 131 at the No. 2 position. In this case, as with dispensing into the dispensing container at the No. 1 position, the drive speed of the liquid feed pump 111 is slowed (e.g., to 15 mL / min) for vials with small dispensing volumes (e.g., 5 mL) to keep the cell concentration variation in the dispensing container 131 within 5%. On the other hand, the drive speed of the liquid feed pump is increased (e.g., to 70 mL / min) for dispensing containers with large dispensing volumes to keep the cell concentration variation in the dispensing container 131 within 5%. (4) After dispensing of the cell solution 117 into the dispensing container at the No. 2 position is completed, the dispensing container sealer unit 130 seals the No. 2 vial. (5) The circuit switching unit 115c at the No. 2 position is switched from an open state to a closed state, and the circuit switching unit 115c at the No. 3 position is switched from a closed state to an open state, and similarly, dispensing is performed for each dispensing container at the No. 3 position and thereafter, and the tubing is sealed after dispensing is completed. (6) When dispensing to all dispensing containers 131 connected via each branch section corresponding to one circuit of the cell fluid circulation circuit unit 110 is completed, the circuit switching unit 115f is switched from an open state to a closed state, and the cell fluid 117 (12) in the circulation circuit 10 is returned to the source bag 118. Note that the above procedure is an example, and dispensing to the dispensing containers 131 is not limited to the above procedure.

[0061] 13 shows the appearance and configuration of the dispensing container sealer unit 130. The role of the dispensing container sealer unit 130 is basically to seal the tubing (connecting portion) of the dispensing container 131 (14) after dispensing. It also serves to seal the tubing and simultaneously insert or cut perforations to separate the dispensing container 131 from the cell solution circulation circuit unit 110.

[0062] The dispensing container sealer unit 130 is composed of an ultrasonic head 132 and an ultrasonic head driver 138. The ultrasonic head 132 is composed of an ultrasonic vibrator 133, an ultrasonic transmission unit 134, and a clamp unit 135 for clamping the tube of the dispensing container 131, which is the object to be sealed. The ultrasonic vibrator 133 is composed of a ceramic laminate and generates ultrasonic waves in a frequency band of 30 to 80 kHz, with output power adjustable. The ultrasonic transmission unit 134 is composed of a material for transmitting ultrasonic waves (metal, ceramic, etc.). Because application of ultrasonic waves significantly accelerates wear, it is often composed of ceramics, carbide, or hardened metal. Meanwhile, the ultrasonic head driver 138 is composed of a motor 136 and a ball screw 137. A servo motor is often used for the motor 136, but a stepping motor or other motor may also be used. The ball screw 137 is used to change the rotational torque of the motor 136 in a linear direction to achieve the desired positioning.

[0063] The operation of dispensing container sealer unit 130 will be described using Figures 13 to 15. As shown in Figure 13, dispensing container sealer unit 130 is installed so that the tube of dispensing container 131 is located in the gap between ultrasonic transmission unit 134 and clamp unit 135. The material of the tube of dispensing container 131 is assumed to be a meltable material such as ethylene vinyl acetate copolymer (EVA) or polyvinyl chloride (PVC). As shown in Figure 14, the tube of dispensing container 131, which is installed in the gap between ultrasonic transmission unit 134 and clamp unit 135 of dispensing container sealer unit 130, is clamped between ultrasonic transmission unit 134 and clamp unit 135. Ultrasonic head driver 138 is driven to press the tube of dispensing container 131, and ultrasonic waves emitted by ultrasonic vibrator 133 are applied to the tube of dispensing container 131 via ultrasonic transmission unit 134, thereby thermally welding the tube.

[0064] FIG. 15 provides a more detailed explanation of the operation of the dispensing container sealer unit 130. As shown in FIG. 13 , the dispensing container sealer unit 130 is initially positioned so that the tube of the dispensing container 131 is sandwiched between the ultrasonic transmitter 134 and the clamp unit 135, with the tip of the ultrasonic transmitter 134 abutting against the tube of the dispensing container 131. After dispensing into the dispensing container 131 is completed, the clamp unit 135 moves to a position where the protrusion 139 at the tip of the clamp unit 135 abuts against the tube of the dispensing container 131 (a position spaced a distance from the tip of the ultrasonic transmitter 134 equivalent to the tube height (outer diameter)), as shown in the diagram in the upper left corner of FIG. 15 . From this tube height position, the movement of the clamp unit 135 switches from position control to load control. During position control, the clamp unit 135 is nearly unloaded, but if the clamp unit 135 is further pushed in from the tube abutment position by load control, the current value applied to the motor increases. The increase in the current value is detected, and when the current value exceeds a predetermined load, the ultrasonic head driver 138 is stopped and controlled to maintain the stopped position. As a guide, the predetermined load is approximately 35 N per tube for ethylene vinyl acetate copolymer (EVA) and approximately 20 N per tube for polyvinyl chloride (PVC).

[0065] When a predetermined load is reached through load control, as shown in FIG. 15 , ultrasonic transducer 133 is driven to start oscillating ultrasonic waves, applying ultrasonic energy to the tube of dispensing container 131 to melt the tube. The ultrasonic oscillation conditions in this case are typically a frequency f of 35 kHz, an amplitude P of 20-25 μm, and an emission time T of approximately 300 milliseconds. For ultrasonic waves, a frequency f of 70 kHz may be used to achieve even greater welding stability. When the tube of dispensing container 131 melts, the load value momentarily drops. Capturing this fluctuation and stopping the ultrasonic emission can improve the joining stability of the tube weld.

[0066] Figure 16 illustrates the exterior and configuration of the dispensing container decant unit 140. The role of the dispensing container decant unit 140 is to stabilize the quality of the dispensed cell solution by preventing cell precipitation and aggregation. Cells in the cell solution 117 contained in the dispensing container 131 precipitate and aggregate after a certain period of time. Cell aggregation can cause respiratory distress, so preventing aggregation is essential. As shown in Figure 16, the dispensing container decant unit 140 is composed of a dispensing container gripper 142 that grips the dispensing container holder 141 and a dispensing container driver 143 that swings the dispensing container holder 141. The dispensing container driver 143 can rotate ±180° and is driven when the cells in the dispensing container precipitate and aggregate. The dispensing container gripper 142 grips the dispensing container holder 141 and is capable of maintaining a constant temperature (0°C to 10°C). Furthermore, the dispensing container holder 141 is detachable from the dispensing container gripping portion 142, and can be directly transferred to the next step, the cryopreservation step.

[0067] FIG. 17 illustrates the decant timing of the dispensing container decant unit 140, and FIG. 18 illustrates the state of suspension of cells in the cell solution in the dispensing container 131 when decanting the dispensing container 131 at such timing. As shown in FIG. 17 , in the initial state (a short time after dispensing), with a decant angle of 0°, the dispensing container 131 faces upward. In this state, as shown in FIG. 18 (0 minutes), cells are aggregated at the bottom of the dispensing container 131. Following the initial state, the decant angle is rotated 180° over approximately 15 seconds. After the 180° rotation, the dispensing container 131 faces downward (inverted). This state is maintained for one minute. Then, due to gravity and shaking over time, the cells in the cell solution 117 in the dispensing container 131 change from aggregated to partially dispersed, as shown in FIG. 18 (1 minute). Next, the decant angle is rotated -180° over approximately 15 seconds. After being rotated by -180°, the dispensing container 131 is again facing upward. This state is maintained for 30 seconds. Then, as time passes, gravity and rocking cause the cells in the cell solution 117 in the dispensing container 131 to change from a partially dispersed state to a more dispersed state, as shown in FIG. 18 (0.5 minutes). Next, the decanting angle is rotated by 180° again over approximately 15 seconds, and the dispensing container 131 is maintained in this state for one minute. Then, as time passes, gravity and rocking cause the cells in the cell solution 117 in the inverted dispensing container 131 to become almost uniformly dispersed, as shown in FIG. 18 (second time, one minute). Next, the decanting angle is rotated by -180° again over approximately 15 seconds, causing the dispensing container 131 to face upward again, and the dispensing container 131 is maintained in this state for one minute. Then, as time passes, due to gravity and rocking, the cells in the cell solution 117 in the dispensing container 131 are maintained in an almost uniformly dispersed state, as shown in FIG. 18 (second time, 0.5 minutes).

[0068] Figure 19 illustrates the exterior and configuration of the dispensing container cooling unit 150. The role of the dispensing container cooling unit 150 is to maintain the cell solution temperature between 4°C and 8°C during dispensing, because if the temperature of the cell solution 117 is close to room temperature, the cells contained therein will become activated and consume more oxygen, resulting in cell aggregation and the presence of cryopreservation solution affecting cell quality. The dispensing container cooling unit 150 is composed of a cooling head unit 151 and a cooling head drive unit 155. The cooling head unit 151 is composed of a Peltier element 152, a cooling plate (metal, ceramic, gel resin, etc.) 153 that transmits the temperature of the Peltier element 152, and a heat insulator 154 to prevent condensation. The cooling head drive unit 155 is a vertical movement mechanism that enables the rotation of the cooling head unit 151 and is composed of a motor 156 and a cam 157 directly connected to it. It also has a temperature sensor 158 that measures the surface temperature of the dispensing container 131.

[0069] FIG. 20 shows the relationship between the temperature of the dispensing container 131 and the decanting timing. First, the current of the Peltier element 152 is controlled to set the temperature of the cooling plate 153 to 2°C. Next, the cooling plate 153 is brought into contact with the dispensing container holder 141 at the starting point of the vertically moving cooling plate 153. The system then waits until the temperature of the dispensing container 131 in the dispensing container holder 141 reaches a predetermined temperature (4°C). To shorten the waiting time, the dispensing container holder 131 can be cooled to the predetermined temperature in advance. If this still requires time, the current value of the Peltier element 152 can be increased to speed up the cooling. After the temperature sensor stabilizes at the set value (4°C), the dispensing container holder 141 is removed from the cooling plate 153 for decantation. In this case, the temperature of the dispensing container 131 is kept within the range of 4°C to 7°C, even including the decanting time (approximately 4 minutes). Continuous monitoring by the temperature sensor 158 may be performed to issue an alarm in the event of an abnormality.

[0070] The technology of the present disclosure primarily relates to a method for evenly dispensing a cell liquid contained in a receiving liquid container (such as a source bag) into a plurality of dispensing containers (such as vials), and a dispensing device for carrying out this method. However, since the liquids dispensed into each dispensing container and individually sealed are cryopreserved as a cell preparation and used in regenerative medicine, etc., the present disclosure can also be said to provide a method and device for manufacturing a cell preparation.

[0071] Furthermore, the technology disclosed herein is primarily applicable to the field of regenerative medicine, which deals with cells, but in the future it can also be utilized in fields that use closed circuits (such as the fields of drug solutions and drug discovery).

[0072] The technology of the present disclosure is not limited to the above-described embodiments, and various modifications and variations are possible without departing from the spirit and scope of the technology of the present disclosure. Therefore, the following claims are appended to disclose the scope of the technology of the present disclosure.

[0073] This application claims priority based on Japanese Patent Application No. 2024-104792, filed on June 28, 2024, the entire contents of which are incorporated herein by reference.

[0074] 10 Circulation circuit 11 Cell 12 Cell fluid 13 Valve (opening / closing valve) 14 Dispensing container (vial) 15 Liquid delivery pipeline (circuit piping section) 20 Source bag body 21 Upper insertion tube 22 Lower discharge tube 23 Source bag outer peripheral gripping section 24 Coating agent 25 Tube cell fluid diffusion section 26 Slit 31 Filter 32a Individual container IN tube 32b Individual container OUT tube 33 Joint 41 Bag 100 Dispensing device 100a Main frame (support base) 100b Main plate (vertical base) 110 Cell fluid circulation circuit section 111 (a, b) Liquid delivery pump 112 Flow rate measurement section 113 Circuit piping section (circulation circuit + branch circuit) 114 Circuit holder section 115 (a, b, c, d, e, f) Circuit switching unit (three-way stopcock, on-off valve, etc.) 116 Cell liquid agitation unit 117 Cell liquid 118 Source bag (container for liquid to be dispensed) 119 Source bag cooling unit 120 (a, b) Air bubble detection unit 121 (a, b, c) Filter 122 Pressure sensor 123 Sterile connector 130 Dispensing container sealer unit 131 Dispensing container 132 Ultrasonic head unit 133 Ultrasonic vibrator 134 Ultrasonic transmission unit 135 Clamp unit 136 Motor 137 Ball screw 138 Ultrasonic head drive unit 139 Convex portion at tip of clamp unit 140 Dispensing container decant unit 141 Dispensing container holder 142 Dispensing container gripping unit 143 Dispensing container drive unit 150 Dispensing container cooling unit 151 Cooling head unit 152 Peltier element 153 Cooling plate 154 Heat insulating material 155 Cooling head driving unit 156 Motor 157 Cam 158 Temperature sensor 160 Cell liquid mixing and stirring unit 161 Sauce bag temperature detection unit 162 Sauce bag cooling unit 163 Sauce bag swing driving unit 164 Sauce bag gripping unit 165 Sauce bag up / down driving unit 166 Sauce bag up / down driving support base 167 Sauce bag flow rate detection unit

Claims

1. A flow path preparation step for preparing a flow path system having a circulation circuit consisting of a piping line connected to a liquid receiving container having a liquid withdrawal port and a liquid return port containing a cell liquid, the piping line extending from the liquid withdrawal port to the liquid return port, a liquid delivery pump provided midway through the circulation circuit, and a plurality of branch circuits each consisting of piping lines branching off via a circuit switching unit connected to the circulation circuit between the outlet of the liquid delivery pump and the liquid return port, with a dispensing container connected to the tip of each branch circuit, and a dispensing flow path from the liquid receiving container to each dispensing container and a circulation flow path returning to the liquid receiving container being individually formed by operating the circuit switching unit; an initial circulation step for operating the circuit switching unit and driving the liquid delivery pump while continuously or intermittently stirring the cell liquid in the liquid receiving container, thereby creating a state in which the cell liquid in the liquid receiving container is withdrawn and flows through the circulation circuit and returned to the liquid receiving container; A dispensing method characterized by comprising: an initial dispensing step, following the initial circulation step, in which the circuit switching unit and the liquid delivery pump are operated to create a state in which the cell liquid is delivered to a dispensing container connected to the tip of one of the plurality of branch circuits; and at least one subsequent dispensing step, after completion of the initial dispensing step, in which the circuit switching unit and the liquid delivery pump are operated to create a state in which the cell liquid is delivered to a dispensing container connected to the tip of another of the plurality of branch circuits.

2. The dispensing method according to claim 1, wherein at least one of the subsequent dispensing steps includes, before the step is performed, operating the circuit switching unit to create a circulating state in which the cell liquid in the dispensing liquid container is extracted, flows through the circulation circuit, and is returned to the dispensing liquid container.

3. The dispensing method according to claim 1 or 2, wherein at least one of the initial dispensing step and the subsequent steps includes an operation of continuously or intermittently stirring the cell liquid in the container for receiving the liquid.

4. A dispensing method according to any one of claims 1 to 3, wherein at least one of the initial circulation step and the subsequent steps includes an operation of circulating the cell liquid in the dispensing liquid container through a path separate from the circulation circuit.

5. A dispensing method according to any one of claims 1 to 4, wherein at least one of the initial circulation step and the subsequent steps is controlled to suppress fluctuations in the surface temperature of the container for the liquid to be dispensed.

6. The dispensing method according to any one of claims 1 to 5, wherein the initial dispensing step and the subsequent dispensing step include an operation of detecting the amount of cell liquid in the dispensing container by measuring the liquid delivery speed of the liquid delivery pump in real time.

7. A dispensing method according to any one of claims 1 to 6, wherein the liquid delivery speed of the liquid delivery pump in the first circulation step is higher than the liquid delivery speed of the liquid delivery pump in the first dispensing step and the subsequent dispensing steps.

8. A method for producing a cell preparation, comprising: storing a cell liquid in each dispensing container using the dispensing method described in any one of claims 1 to 7; welding or cutting the connection between each dispensing container and the branch circuit after dispensing; and freezing and storing each welded or cut dispensing container.

9. The manufacturing method according to claim 8, further comprising vibrating or shaking each of the dispensing containers after a predetermined time has elapsed since dispensing into each of the dispensing containers has been completed.

10. The manufacturing method according to claim 8 or 9, which includes controlling the surface temperature of each dispensing container after dispensing to suppress fluctuations.

11. A dispensing device comprising a circulation circuit consisting of a pipe extending from an inlet end to an outlet end, a liquid delivery pump provided midway through the circulation circuit, and a plurality of branch circuits consisting of pipes each connected via a circuit switching unit to the circulation circuit between the outlet of the liquid delivery pump and the outlet end, wherein the liquid withdrawal port and liquid return port of a liquid container containing cell liquid are connected to the inlet end and outlet end of the circulation circuit, respectively, and a dispensing container is connected to each end of the plurality of branch circuits, and a flow path system configured so that by operating the circuit switching unit and the liquid delivery pump, a dispensing flow path from the liquid container to each dispensing container and a circulation flow path returning to the liquid container can be individually and selectively formed, and a liquid stirring means for shaking or pressing the liquid container connected to the circulation circuit to stir the cell liquid in the liquid container.

12. The dispensing device according to claim 11, wherein the container is a sauce bag, and the liquid stirring means includes a mechanism for pressing the sauce bag.

13. The dispensing device according to claim 11 or 12, further comprising a separate circulation circuit for extracting the cell liquid from the container and returning it to the container via a path separate from the circulation circuit.

14. A dispensing device as claimed in any one of claims 11 to 13, comprising a measuring means for measuring the surface temperature of a container of liquid to be dispensed connected to the circulation circuit, and a control means for controlling the surface temperature to suppress fluctuations based on the results measured by the measuring means.

15. A dispensing device according to any one of claims 11 to 14, comprising a monitoring means for measuring the flow rate of the cell fluid flowing through the circulation circuit in real time and monitoring the amount of cell fluid sent into each dispensing container based on the measurement results.

16. A manufacturing device for a cell preparation, comprising the dispensing device according to any one of claims 11 to 15, and welding or cutting means for welding or cutting the connection between the dispensing containers connected to the respective ends of the plurality of branch circuits and the branch circuits.

17. The manufacturing apparatus according to claim 16, further comprising a vibration means or a shaking means for shaking or vibrating each of the dispensing containers.

18. A manufacturing apparatus according to claim 16 or 17, further comprising means for measuring the surface temperature of each dispensing vessel and controlling the surface temperature of the dispensing vessel to suppress fluctuations based on the measurement results.

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