Cell enrichment apparatus and cell enrichment method

Abstract

To efficiently concentrate rare cells in biological samples. [Solution] The cell concentration apparatus according to the embodiment comprises a first selection unit, a culture unit, and a second selection unit. The first selection unit selects a first cell population from among a plurality of cells contained in a liquid sample, consisting of multiple types of cells whose cell size is greater than or equal to a first threshold. The culture unit expands and cultures the target cells to be concentrated, which are contained in the selected first cell population. The second selection unit selects a second cell population from the expanded and cultured first cell population, consisting of multiple types of cells whose cell size is greater than or equal to a second threshold (larger than the first threshold), and which also contains the target cells.

Description

Technical Field

[0001] The embodiments disclosed in this specification and the drawings relate to a cell concentration device and a cell concentration method.

Background Art

[0002] In recent years, induced pluripotent stem cells (iPS cells) have attracted attention. In order to establish iPS cells, a step of introducing reprogramming factors is required. As cells targeted for this factor introduction, CD34-positive cells, which are hematopoietic progenitor cells, are known. Therefore, if a large amount of CD34-positive cells are contained in the blood used as a material for iPS cells, the reprogramming factors can be introduced efficiently.

[0003] However, generally, CD34-positive cells are rare cells, and it is known that there is only about one CD34-positive cell per one million blood cells. Therefore, a technique for efficiently concentrating CD34-positive cells in blood is required.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to efficiently concentrate rare cells in a biological sample. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problems. The problems corresponding to the respective effects of each configuration shown in the embodiments described later can also be regarded as other problems.

Means for Solving the Problems

[0006] The cell enrichment apparatus according to this embodiment comprises a first sorting unit, a culture unit, and a second sorting unit. The first sorting unit sorts a first cell population consisting of multiple types of cells whose cell size is greater than or equal to a first threshold from among a plurality of cells contained in a liquid sample. The culture unit expands and cultures the target cells to be enriched, which are contained in the sorted first cell population. The second sorting unit sorts a second cell population from the expanded and cultured first cell population consisting of multiple types of cells whose cell size is greater than or equal to a second threshold (larger than the first threshold), and which also contains the target cells. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 shows an example of a schematic configuration of a cartridge used in a cell production apparatus according to this embodiment. [Figure 2] Figure 2 shows an example of a schematic configuration of a cell production apparatus according to the embodiment. [Figure 3] Figure 3 shows an example of the iPS cell production process (steps) flow realized by the cell production apparatus according to this embodiment. [Figure 4] Figure 4 shows an example of the flow of steps within the blood introduction process, blood cell separation process, and expansion culture process according to the embodiment. [Figure 5] Figure 5 is a block diagram showing an example of the flow path configuration of a cartridge according to this embodiment. [Figure 6] Figure 6 is a diagram illustrating an example of a fluid delivery route in the blood introduction step according to the embodiment. [Figure 7] Figure 7 illustrates an example of a fluid delivery route in the blood filtering step according to the embodiment. [Figure 8] Figure 8 illustrates an example of a liquid delivery route in the PBS rinsing step according to this embodiment. [Figure 9] Figure 9 illustrates an example of a fluid delivery route in the cell retrieval step according to the embodiment. [Figure 10] Figure 10 is a diagram illustrating an example of the liquid delivery path in the first classification step according to the embodiment. [Figure 11] Figure 11 illustrates an example of a fluid delivery route in the expansion culture step according to the embodiment. [Figure 12] Figure 12 is a diagram illustrating an example of the liquid delivery path in the second classification step according to the embodiment. [Figure 13] Figure 13 illustrates an example of the relationship between each step of the iPS cell production process according to the embodiment and the proportion of CD34-positive cells in the sample. [Modes for carrying out the invention]

[0008] The embodiments of the cell enrichment apparatus and cell enrichment method will be described in detail below with reference to the drawings.

[0009] First, the configuration of the cell enrichment device according to this embodiment will be described. Figure 1 is a diagram showing an example of the schematic configuration of the cartridge 10 used in the cell enrichment device. The cartridge 10 consistently carries out all steps of cell production. In this embodiment, the cartridge 10 will be described in particular as a device for producing induced pluripotent stem cells (iPS (induced pluripotent stem) cells).

[0010] As shown in Figure 1, the cartridge 10 of this embodiment is a closed system device comprising a cover 2, piping 3, and a plurality of containers 4a to 4r. The cartridge 10 of this embodiment further includes a filter, a vortex channel, and a drive receiving section, but these are omitted from the explanation in Figure 1.

[0011] Multiple containers 4a to 4r store various liquids used for iPS cell production, as well as liquids generated during the iPS cell production process. The liquids used for iPS cell production are samples and reagents, and specific examples include blood, cell suspension, and physiological saline. As physiological saline, for example, PBS (phosphate-buffered saline) may be used.

[0012] In addition, the sample used in the cartridge 10 is, for example, human blood. Blood is an example of a liquid sample. Among the plurality of containers 4a to 4r, the one in which the sample used for the production of iPS cells is stored is called a sample container, and the one in which the reagent is stored is called a reagent container. A specific example of a sample container is a blood bag. A specific example of a reagent container is a chemical solution bag.

[0013] Note that the sample is not limited to blood, and other cell suspensions or the like may also be used. The liquid flowing through the flow path in the cartridge 10 of the present embodiment is also referred to as a fluid.

[0014] Among the plurality of containers 4a to 4r, those other than the sample container and the reagent container such as a blood bag and a chemical solution bag are called liquid storage containers. Specific examples of liquid storage containers include an intermediate container and a recovery container. Hereinafter, when the individual containers 4a to 4r are not particularly distinguished, they are simply referred to as container 4.

[0015] The plurality of pipes 3 are, for example, pipe members through which various liquids used for the production of iPS cells flow. For the pipe 3, for example, a silicone tube is used.

[0016] The pipe 3 and the container 4 constitute a flow path 300 for feeding the liquid used for the production of iPS cells in the cartridge 10. In the cartridge 10 of the present embodiment, the flow path 300 is a closed system flow path. Therefore, the flow path 300 of the cartridge 10 prevents the leakage of liquid and substances to the outside of the flow path 300 and the entry of foreign substances from the outside.

[0017] The cover 2 covers the outside of the cartridge 10. By isolating the inside of the cartridge 10 from the external environment, the cover 2 suppresses the leakage to the outside of the cartridge 10 even if, in the unlikely event, substances such as liquid leak from the closed system flow path inside the cartridge 10. That is, the cartridge 10 has a double-closed structure of the cover 2 and the closed system flow path. The double-closed structure is also referred to as a double-sealing structure.

[0018] Furthermore, cartridge 10 is sterilized and discarded after use, thereby reducing the risk of infection from blood or other substances used in the production of iPS cells. Of the components included in the cell production device 1, cartridge 10 is disposable. For example, cartridge 10 is discarded after each iPS cell production cycle.

[0019] Furthermore, the other components (liquid delivery device 11, valve opening / closing device 12, cooling device 13, incubator 14, and transport device 15) can be used for multiple iPS cell production cycles. Alternatively, the cartridge 10 itself may be reused multiple times after replacing the piping 3, container 4, and filter within the cartridge 10.

[0020] Note that the number, arrangement, and shape of the multiple containers 4a to 4r and pipes 3 shown in Figure 1 are examples only and are not limited to these.

[0021] Figure 2 shows an example of the schematic configuration of the cell production apparatus 1. The cell production apparatus 1 is an example of a cell enrichment apparatus. As shown in Figure 2, the cell production apparatus 1 comprises cartridges 10a, 10b, liquid delivery devices 11a, 11b, valve opening / closing devices 12a, 12b, cooling devices 13a, 13b, incubators 14a, 14b, and transport devices 15a, 15b. The cell production apparatus 1 also includes a processing circuit that controls the entire cell production apparatus 1.

[0022] The liquid transfer devices 11a and 11b drive the transfer of liquid within cartridges 10a and 10b by applying or removing gas pressure to the liquid storage containers of cartridges 10a and 10b.

[0023] For example, the liquid delivery devices 11a and 11b include a regulator for controlling the strength of the gas pressure to be pressurized or depressurized, a pressure pad for transmitting the pressure to the cartridges 10a and 10b, and a moving mechanism for moving the pressure pad to the liquid storage container to be pressurized or depressurized. The liquid delivery devices 11a and 11b may also include a computer or the like for liquid delivery control.

[0024] The valve opening and closing devices 12a and 12b change the liquid delivery path within cartridges 10a and 10b by opening and closing valves provided in the piping of cartridges 10a and 10b.

[0025] Cooling devices 13a and 13b cool the samples and reagents pre-filled in cartridges 10a and 10b to a suitable temperature. Cooling devices 13a and 13b are openable and closable, and cartridges 10a and 10b are inserted into the main body 100 of the cell preparation device 1 by the operator through the openings of the cooling devices 13a and 13b. Before insertion into the cell preparation device 1, cartridge 10 is pre-filled with samples and reagents by the operator.

[0026] Incubators 14a and 14b maintain a suitable environment for cell culture by keeping the temperature, humidity, and carbon dioxide concentration of cartridges 10a and 10b constant. During cell culture, cartridges 10a and 10b are inserted into incubators 14a and 14b by transfer devices 15a and 15b.

[0027] The moving devices 15a and 15b move the positions of cartridges 10a and 10b within the main body 100 of the cell production apparatus 1.

[0028] In the example shown in Figure 2, one cell production apparatus 1 is equipped with two cartridges 10a, 10b, two fluid delivery devices 11a, 11b, two valve opening / closing devices 12a, 12b, two cooling devices 13a, 13b, two incubators 14a, 14b, and two transport devices 15a, 15b. However, this configuration is just one example and is not limited to this.

[0029] For example, the cell production apparatus 1 may be equipped with one of each device, or it may be equipped with three or more devices. Hereinafter, when there is no particular distinction between the two cartridges 10a, 10b, the liquid delivery devices 11a, 11b, the valve opening / closing devices 12a, 12b, the cooling devices 13a, 13b, the incubators 14a, 14b, and the transport devices 15a, 15b, they will simply be referred to as cartridge 10, liquid delivery device 11, valve opening / closing device 12, cooling device 13, incubator 14, and transport device 15.

[0030] The cell production apparatus 1 may also include control devices and other components not shown in Figure 2. The cell production apparatus 1 of this embodiment includes at least a cartridge 10 and a liquid delivery device 11.

[0031] Next, the iPS cell production process performed by the cell production device 1 will be described. iPS cells are cells that are produced by introducing specific genes into somatic cells and then culturing them, and that have the ability to differentiate into cells of various tissues and organs. In this embodiment, nucleated cells from somatic cells, particularly leukocytes, are used as the material for iPS cells.

[0032] Figure 3 shows an example of the iPS cell production process (steps) flow realized by the cell production apparatus 1 according to this embodiment. As shown in Figure 3, the production of iPS cells includes a blood introduction step, a blood cell separation step, a culture expansion step, a reprogramming factor introduction step, a culture step, a subculturing step, and a stock step.

[0033] The blood introduction process involves introducing blood containing white blood cells, which will be used as material for iPS cells, into the cell production device 1.

[0034] The blood cell separation process involves selecting white blood cells from the blood introduced into the cell production device 1, and further extracting only specific cells (hereinafter also referred to as "specific cells"). For example, specific cells are white blood cells whose nuclei are not damaged. In this embodiment, specific cells include CD34 (cluster of differentiation 34) positive cells, which are the target of reprogramming factor introduction.

[0035] The expansion culture process involves increasing the size and proliferation of specific cells contained in the isolated white blood cells.

[0036] The reprogramming factor introduction step is a process of introducing reprogramming factors into specific cells that have been cultured on a large scale. In this embodiment, reprogramming factors are introduced into CD34-positive cells in this step.

[0037] The culture process involves growing cells (iPS cells) into which reprogramming factors have been introduced in a culture vessel.

[0038] The subculturing process involves transferring the iPS cells that have been grown in the culture process to a new culture vessel and continuing to grow them.

[0039] The stocking process is the process of cryopreserving iPS cells that have been subcultured in the subculturing process.

[0040] In the following explanation, each individual process involved in cell production, as shown in Figure 3, will also be referred to as a "step." Each step includes one or more single actions involving the flow of a liquid such as blood or saline solution. In the following explanation, each individual liquid delivery action included in a step will also be referred to as a "step" or "liquid delivery step."

[0041] All the steps shown in Figure 3 are performed by the cell production apparatus 1, but in this embodiment, the blood introduction step, the blood cell separation step, and the expansion culture step will be explained as examples.

[0042] Figure 4 shows an example of the flow of steps within the blood introduction process, blood cell separation process, and expansion culture process according to the embodiment.

[0043] As shown in Figure 4, the blood introduction process includes a blood introduction (blood aspiration) step. The blood cell separation process includes, for example, four steps: a blood filtering step, a PBS rinsing step, a cell recovery step, and a first classification step. The expansion culture process includes, for example, two steps: an expansion culture step and a second classification step.

[0044] Figure 4 illustrates the seven steps included in the blood introduction process, blood cell separation process, and expansion culture process as a continuous flow.

[0045] The first blood introduction step is a fluid delivery operation that introduces the required amount of blood into the pipe 3 of the flow path 300. In other words, the blood introduction step is a fluid delivery operation that takes the required amount of blood from the blood bag into the pipe 3 of the flow path 300, and is therefore also called the blood aspiration step.

[0046] The second blood filtering step involves passing the blood through a filter to separate white blood cells from the blood. Note that the granularity of the separation in the blood filtering step is coarser than in the subsequent first classification step; therefore, the results at this stage may include not only the specific cells being separated but also other blood cells.

[0047] The third PBS rinsing step is the fluid delivery operation in which the cells, which have been filtered, are rinsed with PBS.

[0048] The fourth cell retrieval step is a fluid delivery operation that collects the rinsed cells from the filter.

[0049] The fifth, first classification step is a fluid delivery operation that classifies the cells recovered from the filter. This removes cells other than the specific cells that were not completely removed in the blood filtering step, and extracts the specific cells.

[0050] The sixth expansion culture step is the process of expanding the culture of the extracted specific cells. In this embodiment, this causes the CD34-positive cells that will be targeted for reprogramming factor introduction in the next reprogramming factor introduction step to increase in size and proliferate.

[0051] The seventh, second classification step, is a fluid delivery operation that separates specific cells from the expanded culture. The fluid flow rate differs from that of the fifth, first classification step. This extracts a cell population that contains a large number of CD34-positive cells.

[0052] Here, we will explain the flow path 300 that realizes each step shown in Figure 4.

[0053] Figure 5 shows an example of a flow path configuration that realizes the blood cell introduction process, blood cell separation process, and expansion culture process contained in the cartridge 10 according to the embodiment. The cartridge 10 is an example of a cell concentration device.

[0054] As shown in Figure 5, the flow path 300a includes multiple pipes 3, valves 30a to 30v, filters 51a to 51b, spiral flow paths 52a to 52b, a blood bag 41, a liquid delivery container 42 (42a to 42c), a drug solution bag 43, a recovery container 44, a waste liquid container 45 (45a to 45b), a tapered container 46, a culture medium bag 47, and an expanded culture container 48. Filter 51a and spiral flow path 52a are examples of the first sorting section, and filter 51b and spiral flow path 52b are examples of the second sorting section.

[0055] The blood bag 41, fluid delivery containers 42a-42b, drug solution bag 43, collection container 44, and culture medium bag 47 shown in Figure 5 are examples of containers 4 included in the cartridge 10.

[0056] In this embodiment, the path through which the liquid flows within the flow path 300a due to the liquid delivery operation at each step is called the liquid delivery path. The liquid delivery path differs for each step.

[0057] The blood bag 41 contains blood that will be used as material for iPS cells. The blood bag 41, with the blood inside, is attached to the cartridge 10 by the operator.

[0058] The liquid delivery containers 42a to 42b temporarily store blood or a drug solution such as PBS along the flow path 300a.

[0059] The drug solution bag 43, containing a drug solution such as physiological saline used for iPS cell production, is attached to the cartridge 10 by the operator. In this embodiment, the drug solution bag 43 contains PBS.

[0060] The collection container 44 stores the cells collected from the filter 51a.

[0061] The waste liquid container 45a stores blood cells other than specific cells that have been classified from the cells recovered from the filter 51a.

[0062] The tapered container 46 stores the classified specific cells at the end of the blood cell separation process. The cell suspension containing the cells stored in the tapered container 46 is transferred to the expanded culture vessel 48.

[0063] The culture medium bag 47 contains a chemical solution, such as a culture medium, used for expansion culture. This chemical solution is used to create an environment suitable for culturing the target cells to be concentrated. In this embodiment, the target cells are CD34-positive cells. The culture medium bag 47, with the chemical solution containing the culture medium, is attached to the cartridge 10 by the operator. In this embodiment, the culture medium bag 47 contains a liquid culture medium and cytokines suitable for culturing CD34-positive cells.

[0064] The expanded culture vessel 48 is a culture vessel for expanding the culture of cells contained in the cell suspension delivered from the tapered vessel 46.

[0065] Piping 3 is a tubular member having holes for liquid to flow through. Piping 3 connects filters 51a to 51b, vortex channels 52a to 52b, blood bags 41, liquid delivery containers 42a to 42b, drug solution bags 43, recovery containers 44, waste liquid containers 45a to 45b, tapered containers 46, culture medium bags 47, and expanded culture containers 48.

[0066] As mentioned above, a silicone tube can be used as pipe 3. In this embodiment, the size of pipe 3 is, for example, approximately 3 mm in outer diameter and 1 mm in inner diameter. The connection part of pipe 3 is connected by, for example, a T-joint.

[0067] Valves 30a to 30v are installed in the piping 3 and, under the control of the valve opening / closing device 12, open and close the flow path to change the liquid delivery path within the flow path 300a.

[0068] The following describes each step shown in Figure 4, which is realized by the flow path 300 configured as described above. First, the blood introduction step will be described. As a prerequisite, it is assumed that all valves 30a to 30u are closed before the start of the blood introduction step. Figure 6 is a diagram illustrating an example of the fluid delivery route in the blood introduction step. In Figure 6, the fluid delivery route in the blood introduction step is indicated by (1) and arrows.

[0069] In the blood introduction step, first, the valve opening / closing device 12 opens valve 30a. Then, the fluid delivery device 11 depressurizes the fluid delivery container 42a. This causes blood to be delivered from the blood bag 41 to the fluid delivery container 42a. By depressurizing the fluid delivery container 42a on the inflow side in this way, the fluid delivery device 11 can control the delivery of blood stored in the blood bag 41.

[0070] Next, the blood filtering step will be described. Figure 7 is a diagram illustrating an example of the fluid delivery route in the blood filtering step. In Figure 7, the fluid delivery route in the blood filtering step is indicated by (2) and arrows.

[0071] In the blood filtering step, the valve opening / closing device 12 closes valve 30a and opens valves 30b, 30c, and 30d. Then, the fluid delivery device 11 pressurizes the fluid delivery container 42a. As a result, blood is delivered from the fluid delivery container 42a through the filter 51a to the waste fluid container 45a.

[0072] Filter 51 separates objects from the liquid flowing through channel 300a. Filter 51a is configured to allow particles smaller than separation threshold A to pass through and capture particles larger than or equal to separation threshold A. Separation threshold A is an example of a first threshold. When producing iPS cells from blood, separation threshold A is preferably set to about 6 μm in order to appropriately remove red blood cells and platelets from the blood.

[0073] Next, the PBS rinse step will be described. Figure 8 is a diagram illustrating an example of the fluid delivery route in the PBS rinse step. In Figure 8, the fluid delivery route in the PBS rinse step is indicated by (3) and arrows.

[0074] In the PBS rinsing step, first, the valve opening / closing device 12 opens the valve 30e. Then, the liquid delivery device 11 depressurizes the liquid delivery container 42b. This causes the PBS to be delivered from the chemical bag 43 to the liquid delivery container 42b. By depressurizing the liquid delivery container 42b on the inlet side, the liquid delivery device 11 can control the delivery of the PBS stored in the chemical bag 43.

[0075] After the PBS is delivered to the liquid delivery container 42b, the valve opening / closing device 12 opens the valve 30f. Then, the liquid delivery device 11 pressurizes the liquid delivery container 42b. This causes the PBS to be delivered from the liquid delivery container 42b through the filter 51a to the waste liquid container 45a.

[0076] Next, the cell retrieval step will be described. Figure 9 is a diagram illustrating an example of the fluid delivery route in the cell retrieval step. In Figure 9, the fluid delivery route in the cell retrieval step is indicated by (4) and arrows.

[0077] In the cell retrieval step, first, the valve opening / closing device 12 closes valves 30c, 30d, and 30f. Then, the liquid delivery device 11 depressurizes the liquid delivery container 42b. This causes PBS to be delivered from the drug solution bag 43 to the liquid delivery container 42b.

[0078] After the PBS is delivered to the delivery container 42b, the valve opening / closing device 12 opens valves 30g, 30h, 30i, and 30j. Then, the delivery device 11 pressurizes the delivery container 42b. In the cell retrieval step, PBS is delivered from the delivery container 42b through the filter 51a to the retrieval container 44. As a result, the cells rinsed with PBS are retrieved from the filter 51a and temporarily stored in the retrieval container 44.

[0079] Next, the first classification step will be explained. Figure 10 is a diagram illustrating an example of the liquid delivery route in the first classification step. In Figure 10, the liquid delivery route in the first classification step is indicated by (5) and arrows.

[0080] First, the valve opening / closing device 12 closes valve 30j and opens valves 30l and 30n. Then, the liquid delivery device 11 pressurizes the recovery container 44. As a result, the PBS containing the cells recovered from filter 51ba is delivered from the recovery container 44 through the vortex channel 52a to the waste liquid container 45a and the tapered container 46.

[0081] The vortex channel 52 is an example of a device that sorts objects in a liquid by the action of flow. The vortex channel 52 is also called a helical channel.

[0082] The vortex channel 52a classifies specific cells from the PBS containing cells recovered from the filter 51a based on cell size. The vortex channel 52a is configured to classify particles larger than or equal to separation threshold A by controlling the flow rate. In this case, multiple types of cells classified as particles larger than or equal to separation threshold A are an example of the first cell population.

[0083] In the blood filtering step described above, particles larger than or equal to the separation threshold A are recovered by filter 51a. However, the particle size of the filter is coarser than that of the first classification step using a spiral channel. Therefore, the cells recovered by filter 51a may contain particles smaller than the separation threshold A. In this embodiment, by performing the first classification step, it is possible to more appropriately remove red blood cells and platelets, which are particles smaller than the separation threshold A, from the sample.

[0084] In this embodiment, red blood cells and platelets are removed from the sample by both a filter and a vortex channel. However, red blood cells and platelets may be removed from the sample by either a filter or a vortex channel alone. Furthermore, any method that can select specific cells based on cell size may be used instead of the method described above.

[0085] In this embodiment, cells larger than or equal to the separation threshold A flow into the tapered container 46. Cells smaller than the separation threshold A are discharged into the waste liquid container 45a.

[0086] Furthermore, the valve opening / closing device 12 can also discharge cells classified as particles of size A or larger into the waste liquid container 45a by closing valve 30n and opening valve 30m.

[0087] For example, until a predetermined time has elapsed from the start of fluid delivery, cells classified as particles of size A or larger may be discharged into the waste liquid container 45a, as described above. This allows the classification to stabilize before the cells classified as particles of size A or larger can be flowed into the tapered container 46.

[0088] Next, the expansion culture step will be described. Figure 11 is a diagram illustrating an example of the fluid delivery route in the expansion culture step. In Figure 11, the fluid delivery route in the expansion culture step is indicated by (6)-1, (6)-2, and arrows.

[0089] First, the valve opening / closing device 12 closes valve 30n and opens valves 30o, 30p, and 30q. Then, the liquid delivery device 11 pressurizes the liquid delivery container 42c. This also pressurizes the tapered container 46 connected to the liquid delivery container 42c by the piping 3. As the tapered container 46 is pressurized, PBS containing specific cells is delivered from the tapered container 46 to the filter 51b. The PBS containing particles that have passed through the filter 51b is then delivered to the waste liquid container 45b ((6)-1).

[0090] Here, filter 51b is configured to allow particles below the separation threshold A to pass through and capture particles above the separation threshold A. As a result, cell populations containing specific cells, which are particles above the separation threshold A, are captured by filter 51b, and PBS containing particles below the separation threshold A is moved to waste liquid container 45b.

[0091] After PBS containing particles below separation threshold A is transferred from the tapered container 46 to the waste liquid container 45b, the valve opening / closing device 12 closes valves 30o, 30p, and 30q and opens valve 30r. Subsequently, the liquid transfer device 11 depressurizes the liquid transfer container 42b.

[0092] This allows the culture medium and cytokines to be delivered from the culture medium bag 47 to the liquid delivery container 42c. By reducing the pressure of the liquid delivery container 42c on the inflow side, the liquid delivery device 11 can control the delivery of the culture medium and cytokines stored in the culture medium bag 47.

[0093] After the culture medium and cytokines have been delivered to the liquid delivery container 42c, the valve opening / closing device 12 opens valves 30s and 30t. Then, the liquid delivery device 11 pressurizes the liquid delivery container 42c. As a result, the culture medium and cytokines are delivered from the liquid delivery container 42c through the filter 51b to the expanded culture vessel 48 ((6)-2).

[0094] Therefore, the cell population, including the specific cells captured by filter 51b, is swept away by the culture medium and cytokines and moves to the expanded culture vessel 48 along with the culture medium and cytokines. In other words, the cell population, including the specific cells, is collected in the expanded culture vessel 48 suspended in the culture medium and cytokines by the above-described liquid transfer action.

[0095] In this embodiment, the cell population containing the specific cells collected in the expanded culture vessel 48 is cultured for 5 days. This causes the target cells (target cells) that are the target of reprogramming factor introduction contained in the specific cells to increase in size and proliferate. In this embodiment, CD34-positive cells increase in size and proliferate.

[0096] Next, the second classification step will be explained. Figure 12 is a diagram illustrating an example of the fluid delivery route in the second classification step. In Figure 12, the fluid delivery route in the second classification step is indicated by (7) and arrows.

[0097] First, the valve opening / closing device 12 opens valves 30u and 30v. Then, the liquid delivery device 11 pressurizes the liquid delivery container 42c. This also pressurizes the enlarged culture container connected to the liquid delivery container 42c by the piping 3. As the enlarged culture container 48 is pressurized, the cell suspension (culture medium) containing the enlarged cultured specific cells is divided and delivered from the enlarged culture container 48 through the vortex channel 52b to the waste liquid container 45b and to the piping 3 that leads to the initialization factor introduction unit (not shown) which performs processing related to the initialization factor introduction process.

[0098] The vortex channel 52b classifies cells that are likely to be the target cells from other blood cells in a cell suspension containing enlarged cultured specific cells, based on cell size. The vortex channel 52b is a vortex channel with the same design as the vortex channel 52a, but the flow rate of the fluid delivered during classification differs between the first and second classification steps. Alternatively, the channel 300 may be configured so that the second classification step can be performed using the vortex channel 52a without providing the vortex channel 52b.

[0099] The vortex channel 52a is configured to classify particles larger than or equal to the separation threshold B by controlling the flow rate. In this case, the separation threshold B is an example of a second threshold. Furthermore, multiple types of cells that are classified as particles larger than or equal to the separation threshold B are an example of a second cell population.

[0100] Here, the separation threshold B is a larger value than the separation threshold A. When producing iPS cells from blood, it is preferable to set the separation threshold B to approximately 7-9 μm in order to appropriately remove lymphocytes present in the cell suspension containing the specific cells that have been cultured on a large scale.

[0101] In this embodiment, lymphocytes are removed from the sample using a spiral channel, but lymphocytes may also be removed using a filter. Alternatively, lymphocytes may be removed using both a filter and a spiral channel.

[0102] In this embodiment, cells larger than or equal to the separation threshold B are sent to the piping 3 leading to the reprogramming factor introduction unit. Cells smaller than the separation threshold B are discharged into the waste liquid container 45b.

[0103] By performing the above seven steps, the cell production apparatus 1 according to this embodiment can efficiently concentrate CD34-positive cells. The reason why the cell production apparatus 1 according to this embodiment can efficiently concentrate CD34-positive cells will be explained below with reference to Figure 13. Figure 13 is a diagram illustrating an example of the relationship between each step of the iPS cell production process and the proportion of CD34-positive cells in the sample.

[0104] As shown in Figure 13, the proportion of CD34-positive cells in the sample after the blood introduction step is approximately 1 / 1,000,000. From here, the cell generation device 1 performs a blood filtering step and a first classification step to remove red blood cells and platelets, which are cells below the separation threshold A, from the sample. As a result, the proportion of CD34-positive cells in the sample becomes approximately 1 / 1,000.

[0105] As shown in Figure 13, the cells sorted in the blood filtering step and the first classification step include lymphocytes that are approximately the same size as CD34-positive cells. At this stage, it is not possible to remove lymphocytes that are approximately the same size as CD34-positive cells based on cell size. Therefore, the cell production apparatus 1 according to this embodiment performs the expansion culture step.

[0106] In the expansion culture step, the cell production device 1 cultures the cells selected in the blood filtering step and the first classification step under conditions suitable for CD34-positive cells. As an example, the cell production device 1 expands the culture of multiple cells selected in the blood filtering step and the first classification step for 5 days under conditions in which cytokines are added to StemSpanSFEM (manufactured by Stem Cell Technologies).

[0107] As a result, CD34-positive cells included in the multiple cells sorted in the blood filtering step and the first classification step increase in size and proliferate. Furthermore, since the expansion culture step is performed under conditions suitable for CD34-positive cells, lymphocytes do not increase in size or proliferate. Therefore, lymphocytes that were approximately the same size as CD34-positive cells before expansion culture become smaller than CD34-positive cells after expansion culture.

[0108] Furthermore, granulocytes contained in the cells sorted in the blood filtering step and the first classification step lose their structure when cultured under conditions suitable for CD34-positive cells, and therefore disappear from the sample. In other words, by performing the expansion culture step, granulocytes contained in the cells sorted in the blood filtering step and the first classification step can be removed from the sample. As a result, the proportion of CD34-positive cells in the sample becomes about 1 / 100.

[0109] From here, the cell generation device 1 performs a second classification step to remove lymphocytes, which are cells below the separation threshold B, from the sample. As a result, the proportion of CD34-positive cells in the sample is reduced to about 1 / 10.

[0110] As described above, the cell production apparatus 1 according to this embodiment can remove lymphocytes that were approximately the same size as CD34-positive cells from the sample by performing a second classification step, which is different from the first classification step, after expanding the culture of the cells selected in the blood filtering step and the first classification step.

[0111] The cell production apparatus 1 according to the embodiment described above selects a cell population consisting of multiple types of cells whose cell size is equal to or greater than a separation threshold A from among a plurality of cells contained in a liquid sample, expands the culture of the target cells to be concentrated contained in the selected cell population, and selects a cell population from the expanded cultured cell population whose cell size is greater than a separation threshold A, i.e., a separation threshold B or greater.

[0112] As a result, for example, if the cells to be selected (target cells) are rare cells, the cell production apparatus 1 according to this embodiment can perform expansion culture in an environment suitable for the target cells, thereby increasing the size and proliferation of only the target cells from among multiple types of cells selected as cells meeting or exceeding the separation threshold A. Therefore, the cell production apparatus 1 according to this embodiment can efficiently remove unnecessary cells from the sample that were approximately the same size as the target cells before expansion culture by selecting the cell population that meets or exceeds the separation threshold B after expansion culture. In other words, the cell production apparatus 1 according to this embodiment can efficiently concentrate rare cells in a biological sample.

[0113] In this embodiment, CD34-positive cells are included among the multiple types of cells selected as a cell population whose cell size is greater than or equal to the separation threshold B, as described above. CD34-positive cells are rare cells that serve as targets for introducing reprogramming factors when creating iPS cells from blood. For this reason, conventionally, CD34-positive cells have sometimes been collected using antigen-antibody reactions in order to efficiently introduce reprogramming factors. For example, anti-CD34 antibodies with attached porcelain beads are added to blood and reacted with CD34-positive cells, and then the porcelain beads are attracted with a magnet to collect the CD34-positive cells bound to the porcelain beads via the anti-CD34 antibody. Alternatively, for example, anti-CD34 antibodies with attached fluorescent dyes are added to blood and reacted with CD34-positive cells, and then flow cytometry is used to select and collect the CD34-positive cells bound to the fluorescent dye via the anti-CD34 antibody. However, with the above methods, there is a possibility that unreacted porcelain beads, fluorescent dyes, and other impurities may remain in the sample. Furthermore, it is generally difficult to incorporate a mechanism for collecting CD34-positive cells using the method described above into a cell production device that automatically performs a series of steps such as hematological introduction, hematological separation, expansion culture, reprogramming factor introduction, culture, subculturing, and stocking. In contrast, the cell production device 1 according to this embodiment selects a cell population whose size is equal to or greater than the separation threshold A, and then performs expansion culture in an environment suitable for CD34-positive cells. This allows only CD34-positive cells to be enlarged and proliferated from among multiple types of cells whose size is equal to or greater than the separation threshold A. In addition, by selecting a cell population whose size is equal to or greater than the separation threshold B after expansion culture, lymphocytes whose size was approximately the same as CD34-positive cells before expansion culture can be removed from the sample. In other words, the cell production device 1 according to this embodiment makes it possible to efficiently obtain a sample containing a large number of CD34-positive cells without adding reagents that are not directly related to iPS cell production to the sample or complicating the device configuration.

[0114] The above-described embodiment can also be modified and implemented as appropriate by changing some of the configuration or functions of the cell production apparatus 1. Therefore, the following describes modified examples of the above-described embodiment as other embodiments. In the following, we will mainly describe the differences from the above-described embodiment, and will omit detailed explanations of points that are common with what has already been described. Furthermore, the modified examples described below may be implemented individually or in combination as appropriate.

[0115] (Variation 1) The embodiments described above describe a method for concentrating CD34-positive cells in the blood. However, the target cells to be concentrated may be cells other than CD34-positive cells. The target cells can be any type of cell that, when cultured on a larger scale, will produce a size difference between them and other cells.

[0116] According to this modified method, it is possible to efficiently enrich rare cells other than CD34-positive cells.

[0117] (Modification 2) In the above-described embodiment, a configuration was explained in which a series of processes related to the production of iPS cells, including the blood introduction process, blood cell separation process, expansion culture process, reprogramming factor introduction process, culture process, subculturing process, and stocking process, are performed in a single cell production apparatus 1. However, these processes may be performed in multiple apparatuses. In this case, each process may be handled by a different apparatus, or there may be an apparatus that handles multiple processes.

[0118] In the above-described embodiment, we have explained a configuration in which the cell production apparatus 1 automatically performs all of the processes related to the above steps, but some or all of the processes related to the above steps may be performed by a human.

[0119] According to this modified version, rare cells in biological samples can be efficiently concentrated in a manner suitable for the equipment and environment of facilities that produce cells using biological samples as material.

[0120] According to at least one embodiment or modification described above, rare cells in a biological sample can be efficiently concentrated.

[0121] While several embodiments have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These embodiments can be implemented in a variety of other forms, and various omissions, substitutions, modifications, and combinations of embodiments are possible without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims and their equivalents. [Explanation of symbols]

[0122] 1. Cell production device 2 Covers 3,3a,3b Piping 4,4a~4r container 10, 10a, 10b cartridges 11,11a,11b Liquid delivery device 12, 12a, 12b Valve opening and closing device 13,13a,13b Cooling device 14,14a,14b Incubators 15,15a,15b Mobile device 30,30A~30V bulb 41 Blood bags 42, 42a~42c Liquid transfer container 43 Medication bags 44 Collection containers 45, 45a, 45b Waste liquid containers 46 Tapered container 47 Culture bags 48 Enlarged culture vessels

Claims

1. A first sorting unit selects a first cell population consisting of multiple types of cells whose cell size is greater than or equal to a first threshold from among multiple cells contained in a liquid sample, A culture section for expanding the culture of target cells to be concentrated, which are included in the selected first cell population, A second selection unit selects a second cell population from the first cell population that has been cultured on an expanded scale, which consists of multiple types of cells whose cell size is greater than or equal to a second threshold (larger than the first threshold), and which includes the target cells. A cell enrichment device equipped with the following features.

2. The aforementioned liquid sample is blood. The cell enrichment apparatus according to claim 1.

3. The target cells are CD34-positive cells. The first sorting unit sorts the first cell population, which consists of multiple types of cells including the CD34-positive cells, The culture section expands the culture of the CD34-positive cells, The second sorting unit sorts the second cell population, which consists of multiple types of cells including the CD34-positive cells that have been cultured on a large scale. The cell enrichment apparatus according to claim 2.

4. The culture section increases the size and proliferates the target cells by performing large-scale culture in an environment appropriate to the target cells. The cell enrichment apparatus according to claim 1.

5. The first sorting unit is at least one of a filter that allows particles with a size less than the first threshold to pass through and captures particles with a size equal to or greater than the first threshold, and a helical channel that divides particles based on their size. The second sorting unit is at least one of the following: a filter that allows particles with a size less than the second threshold to pass through and captures particles with a size greater than or equal to the second threshold, and the helical flow path. The cell enrichment apparatus according to claim 1.

6. The first sorting unit includes the helical channel and sorts the first cell population by separating particles whose particle size is equal to or greater than the first threshold using a fluid at a predetermined flow rate that flows into the helical channel. The second sorting unit includes the helical channel, and sorts the second cell population by using the helical channel and a fluid flow rate different from the predetermined flow rate to separate particles whose particle size is equal to or greater than the second threshold. The cell enrichment apparatus according to claim 5.

7. A first selection step involves selecting a first cell population from among multiple cells contained in a liquid sample, consisting of multiple types of cells whose cell size is greater than or equal to a first threshold, A culture step of expanding the culture of target cells to be enriched, which are included in the selected first cell population, A second selection step involves selecting a second cell population from the first cell population that has been cultured on a large scale, which consists of multiple types of cells whose cell size is greater than or equal to a second threshold (larger than the first threshold), and which includes the target cells. A cell enrichment method including the following.

8. The aforementioned liquid sample is blood. The cell enrichment method according to claim 7.

9. The target cells are CD34-positive cells. The first selection step selects the first cell population consisting of multiple types of cells, including the CD34-positive cells, The culture step involves expanding the culture of the CD34-positive cells, The second selection step involves selecting the second cell population, which includes the expanded cultured CD34-positive cells. The cell enrichment method according to claim 8.

10. The culture step involves performing an expanded culture in an environment appropriate to the target cells to increase their size and proliferate them. The cell enrichment method according to claim 7.

11. The first sorting step sorts the first cell population using at least one of a filter that allows particles with a size less than the first threshold to pass through and captures particles with a size greater than or equal to the first threshold, and a helical channel that divides particles based on their size. The second sorting step involves sorting the second cell population using at least one of a filter that allows particles with a size less than the second threshold to pass through and captures particles with a size greater than or equal to the second threshold, and the helical channel. The cell enrichment method according to claim 7.

12. The first sorting step involves controlling the flow rate of the fluid introduced into the helical channel and separating particles whose size is greater than or equal to the first threshold, thereby sorting the first cell population. The second sorting step uses the helical channel used in the first sorting step, and differs from the first sorting step in that it separates particles whose particle size is equal to or greater than the second threshold, thereby sorting the second cell population. The cell enrichment method according to claim 11.

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