WO2020196649A1 - Cell production device, cell production method, and server, system and device to be used therefor - Google Patents

Abstract

To provide a cell production device, etc. whereby the qualities of target cells to be collected and yield thereof can be reproducibly improved. A device for producing a product containing target cells, said device comprising: a means for discharging a target cell-containing starting liquid material to a cell filtration filter; a means for collecting the target cell-containing filtrate obtained by the cell filtration filter; and a means for variably regulating the operating conditions of the coordinates for the filter that conducts the discharge. Preferably, this device further comprises a means for variably regulating the operation conditions of the discharge speed.

Description

Cell manufacturing equipment, cell manufacturing methods, and servers, systems and equipment used for them.

The present invention relates to a cell manufacturing apparatus, a cell manufacturing method, and a server, a system, and an apparatus used therein.

The treatment of collecting cells from the body fluids and tissues of the patient or the donor, amplifying and processing them in culture, and transplanting them to the affected area, so-called regenerative medicine and cell medicine, is for diseases that are difficult to treat with conventional technology. It has the potential to provide new treatments and is attracting attention. (See, for example, Non-Patent Document 1.). In addition to the above, we also conduct drug discovery research for personalized medicine that collects cells from the body fluids and tissues of the patient or the donor, elucidates the cause of the disease from the cells amplified and processed by culturing, and develops new drugs. It is receiving a lot of attention.

The manufacturing process of products containing cells used in drug discovery research for regenerative medicine / cell medicine and personalized medicine described above spans multiple steps. For example, a step of collecting cells from a tissue or the like, a step of culturing and amplifying the obtained cells (expansion culture), a step of culturing cells and processing them into target cells (differentiation induction culture), etc. can be mentioned. Also in the process, a step of collecting the target cells is required.
For example, in the step of collecting cells from a tissue or the like, it is necessary to remove impurities contained in the tissue from the tissue. Further, it is known that during differentiation-inducing culture, pluripotent stem cells such as ES cells and iPS cells induce differentiation into various cells while forming cell aggregates over time. Therefore, in order to recover high-purity target cells, other cells, cell clusters, cell debris (also referred to as cultured debris), impurities, etc. generated during culture are removed from the target cells, and the target cells are removed. Only need to be recovered.
Generally, such a recovery operation is performed by filtering the cell suspension with a flat membrane type cell filtration / separation device such as a filtration filter.

On the other hand, in recent years, in order to efficiently produce products containing cells, devices have been developed that mechanize from culture operations such as cell seeding and medium exchange to formulation and commercialization.
For example, in Patent Document 1, a factor introduction device, a cell mass preparation device, an initialization culture device, an expansion culture device for expanding and culturing a plurality of cell clusters, and a medium supply device for supplying a medium are provided to establish stem cells. A manufacturing apparatus that packages from to expanded culture is disclosed.

Japanese Unexamined Patent Publication No. 2017-22187

In Patent Document 1, a filter is provided in the liquid feeding path for the purpose of removing dead cell clumps. However, when the dead cell mass is removed from the culture solution and only the target cells are to be separated by the filter provided in the liquid feeding path, the culture solution is applied to the entire surface of the filter at once, so that the inside of the filter is used. Cultured debris, contaminants, and dead cell clumps are deposited and clogged. Therefore, it is not possible to efficiently recover only the target cells, and the recovery rate and recovery efficiency are significantly lowered. Further, if the feeding rate of the culture solution is increased in order to improve the recovery rate, the damage when the filter and the cells collide with each other increases, cell death occurs, and the quality of the recovered target cells deteriorates.
Therefore, it is an object of the present invention to provide a cell production apparatus, a cell production method, and a server, a system, and an apparatus used for the cell production apparatus and the cell production method for improving the recovery rate and quality of the target cells to be recovered.

As a result of diligent studies and researches to solve the above problems, the present inventors have found that the coordinates of the filter for discharging the raw material liquid to the cell filtration filter affect the viability and yield of the target cells. The present invention has been completed.

That is, the present invention that solves the above problems is an apparatus for producing a product containing a target cell, which is a means for discharging a raw material liquid containing the target cell to a cell filtration filter and a target cell obtained by the cell filtration filter. It is a device including means for collecting the filtered liquid containing the above-mentioned material and means for variably regulating the operating conditions of the coordinates with respect to the filter for discharging.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, an apparatus further comprising means for variably regulating the operating conditions of the discharge rate is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, an apparatus further comprising means for variably regulating the liquid passing time of the cell filtration filter is shown.

In one embodiment of the apparatus for producing a product containing the target cells according to the present invention, one or more additional operating conditions selected from the group consisting of the number of times of the discharge, the number of the means for discharging and the discharge pressure. A device further comprising means for variably or non-variably regulating.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, the operating conditions are variable, and the apparatus detects the number of cells contained in the filtered liquid and based on the detection. A device further comprising means for adjusting the operating conditions is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, the operating conditions are variable, and the apparatus causes the accumulation of filters in coordinates with respect to the discharging filter or an event correlating therewith. An apparatus further comprising a means for detecting and a means for adjusting the operating condition based on the detection is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, an apparatus further comprising means for attaching the cell filtration filter to the opening of a container for collecting the filtered liquid is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, the mounting means is a means for gripping two or more of the cell filtration filters or a support for supporting the container, and the gripping means. A device comprising a means of transporting a support and aligning the two or more cell filtration filters with an opening of the container is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, an apparatus further comprising means for removing the cell filtration filter from the opening of a container for collecting the filtered liquid is shown.

In one embodiment of an apparatus for producing a product containing a target cell according to the present invention, an apparatus further comprising a means for recovering the target cell by solid-liquid separation from the filtered liquid is further shown.

In one embodiment of the apparatus for producing a product containing the target cells according to the present invention, the solid-liquid separation is centrifugal separation, and the collecting means is a means for closing the opening of the container in which the filtered liquid is collected. And a device comprising means for applying centrifugal force to the container.

The present invention that solves the above-mentioned problems is a method for manufacturing the above-mentioned product by using the above-mentioned apparatus for manufacturing the product containing the target cell.

The present invention, which solves the above-mentioned problems, refers to a database that stores data relating to the correspondence between the coordinates of the discharging filter and the recovery rate of the target cells in the product, and the required recovery by referring to the database. A server including means for selecting operating conditions of coordinates for the filter performing the ejection for achieving the rate, and means for transmitting information on the selected operating conditions to the device for manufacturing a product containing the target cells described above. Is.

In one embodiment of the server according to the invention, the database further stores data relating to the correspondence between the discharge rate and the viability of the target cell in the product, and the means of selection is the required said. The means for selecting and transmitting the operating conditions of the discharge rate for achieving the survival rate is indicated by a server that transmits the information to the device for manufacturing the product containing the target cells described above.

The present invention that solves the above-mentioned problems is a system including the above-mentioned apparatus for manufacturing a product containing a target cell and the above-mentioned server.

In one embodiment of the system, the apparatus manufactures the product based on operating conditions of information transmitted from the server, resulting in the rate of discharge and the survival rate of the target cells in the product. The actual data regarding the correspondence relationship with the server or the correspondence relationship between the coordinates for the filter performing the discharge and the recovery rate of the target cells in the product is transmitted to the server, and the database additionally stores the actual data. The system to do is shown.

In one embodiment of the above apparatus, the required recovery is performed with reference to a database that stores data relating to the correspondence between the coordinates of the discharging filter and the recovery rate of the target cells in the product, and the database. A device further provided with means for selecting the operating conditions of the coordinates for achieving the rate and for the discharging filter is shown.

In one embodiment of the apparatus, the database further stores data relating to the correspondence between the rate of discharge and the viability of the target cell in the product, and the means of choice is the required survival. An apparatus is shown which selects the operating conditions of the discharge rate to achieve the rate.

According to the present invention, the working efficiency is maximized while improving the survival rate and recovery rate of the target cells by variably regulating the operating conditions of the coordinates for the filter that discharges the raw material liquid to the cell filtration filter. be able to.

It is a perspective view of the cell production apparatus which concerns on one Embodiment of this invention. It is a figure which shows typically the state and operation at the time of discharging a raw material liquid on a cell filtration filter. In one embodiment of the cell manufacturing apparatus of the present invention, it is a figure schematically showing an example of a place on a filter to which a raw material liquid is applied, corresponding to coordinates with respect to a filtration filter from which the raw material liquid is discharged. It is a figure which shows the flow of cell production which concerns on one Embodiment of this invention. It is a figure which shows the flow of cell production which concerns on one Embodiment of this invention.

Hereinafter, a detailed description will be given based on the embodiment of the present invention.

(Cell manufacturing equipment)
The cell manufacturing apparatus according to the present invention is an apparatus for producing a product containing target cells, and includes means for discharging a raw material liquid containing target cells to a cell filtration filter and target cells obtained by the cell filtration filter. A means for collecting the filtered liquid and a means for variably regulating the operating conditions of the coordinates with respect to the filter for discharging the filtered liquid are provided.

In the present specification, the "coordinates" with respect to the filter for discharging means the position of the place where the raw material liquid is discharged from the discharging means relative to the filter. Simply, when the first axis in the plane when the filter is viewed in a plane is the X axis and the second axis in the plane intersecting the first axis is the Y axis, the X-axis coordinates and the Y-axis coordinates are used. It may include the coordinates represented. Further, the coordinates may include the distance between the place where the raw material liquid is discharged by the discharging means and the surface of the filter (the coordinates of the Z axis which is the orthogonal direction with respect to the plane).

To "variably regulate" the operating conditions of the coordinates, it is sufficient that a plurality of coordinates can be selected and the coordinates do not change randomly without reproducibility, and reproducibility with a width is allowed. Further, it is sufficient if a plurality of coordinates can be set before or during the operation of the device, and the coordinates do not necessarily have to be variable (typically feedback control) during the operation.
Therefore, it suffices that the range of motion of the means for discharging the raw material liquid is restricted mechanically or mechanically by electrical control, and the relative positions can be determined in a plurality of ways.

The cell manufacturing apparatus of the present invention comprises a means for discharging a raw material liquid containing target cells supplied from the upstream side to a cell filtration filter, and a means for collecting a filtered liquid containing target cells obtained by the cell filtration filter. As long as there is a means for variably regulating the operating conditions of the coordinates with respect to the filter that performs the discharge, the mechanism, configuration, etc. before and after that are arbitrary.

For example, the raw material liquid may be prepared manually or in an automatic incubator or the like.

The automatic culture device refers to a device that automatically or semi-automatically performs all or part of the cell culture operation that is normally performed manually by substituting a machine or an instrument. Examples of the cell culture operation include a medium exchange, cell recovery, and washing operation. Specific examples include, for example, Aastrom Replicell System (manufactured by Aastrom Bioscience), and culture devices disclosed by JP-A-2004-344128, JP-A-2004-89095, and JP-A-2001-275569.

FIG. 1 is a perspective view of a cell manufacturing apparatus according to an embodiment of the present invention.
A pipette tip 40 attached to the tip of the dispensing pipette is provided as a means for discharging the raw material liquid containing the target cells to the cell filtration filter 11, a cell filtration filter 11 below the pipette tip 40, and a cell filtration filter 11 below the pipette tip 40. Is provided with a container 30 as a means for collecting the filtered liquid containing the target cells.

A dispensing head 42 communicated with a raw material liquid source (not shown) is fixed to a head drive device 70 (Z-axis drive mechanism 73 in this figure) including an X-axis drive mechanism 71, a Y-axis drive mechanism 72, and a Z-axis drive mechanism 73. Further, the pipette tip 40 is detachably fixed to the tip of the dispensing head 42. Further, the cell filtration filter 11 is installed parallel to the planes of the X-axis and the Y-axis, and horizontally in this embodiment.

Here, since the X-axis drive mechanism 71 and the Y-axis drive mechanism 72 are slidably connected to each other, the trajectory of the tip of the pipette tip 40 and the relative position of the tip of the pipette tip 40 with respect to the filter are restricted to be reproducible. Will be done. As a result, the operating conditions of the coordinates with respect to the filter to which the raw material liquid naturally falls from the pipette tip 40 are variably regulated. As described above, in the present embodiment, the X-axis drive mechanism 71 and the Y-axis drive mechanism 72 correspond to the regulatory means.

The means for discharging the raw material liquid containing the target cells to the cell filtration filter 11 is not limited to the above-mentioned dispensing pipette, and an appropriate discharging means may be used according to the size of the filter or the container for collecting the filtrate. , Pipettes, micropipettes, syringes and the like. The diameter of the discharge portion of the discharge means is not particularly limited as long as the raw material liquid can be discharged, but the inner diameter is, for example, 0.03 mm or more, 0.05 mm or more, 0.15 mm or more, 0.56 mm or more as the lower limit. It is preferably 1.00 mm or more and 2.50 mm or more, and the upper limit is preferably 8.00 mm or less, 5.00 mm or less, and 4.00 mm or less.

FIG. 2 schematically shows a state and an operation when the raw material liquid is discharged onto the cell filtration filter. When the raw material liquid is discharged from the pipette tip 40 onto the cell filtration filter 11, the filtered liquid containing the target cells that have passed through the cell filtration filter 11 is collected in the container 30 (a). At this time, the cell filtration filter 11 is basically designed to allow cells of interest to pass through, while filtering out other cells, cell clumps, debris, impurities, etc. without passing through. However, as the amount of the raw material liquid discharged to a certain place on the filter 11 increases, the filter medium 5 at the place of the filter 11 corresponding to the coordinates becomes larger and begins to trap the target cells to be passed (b). The present inventor has found that.
This trap phenomenon is a factor that reduces the recovery rate of target cells. In the present invention, by varying the relative position of the pipette tip 40 with respect to the filter 11, the discharge amount of the raw material liquid per place of the filter 11 is reduced, and the trap phenomenon is suppressed. Further, by restricting the variability of the relative position to be reproducible and variably restricting the operating conditions of the coordinates with respect to the filter 11 for discharging, the trap phenomenon is suppressed reproducibly and the recovery rate of the target cells is improved. Let me.

As the number of coordinates increases, the amount of raw material liquid applied per coordinate decreases and the recovery rate of target cells improves. On the other hand, when the number of coordinates is large, there may be disadvantages that the number of raw material liquid discharging means is increased and the operating conditions are complicated because the position of the discharging means is reproducibly changed. Therefore, the method of variably regulating the operating conditions of the coordinates with respect to the filter is not particularly limited, and may be appropriately set by comprehensively considering the recovery rate of the target cells and the simplicity of the device configuration and operating conditions. ..

FIG. 3 schematically shows an example of a location on the filter to which the raw material liquid is applied, corresponding to the coordinates with respect to the filtration filter from which the raw material liquid is discharged in one embodiment of the cell manufacturing apparatus of the present invention. In FIGS. 3 (b) to 3 (k) and FIG. 2 (c) described above, the solid circles are virtual marks for illustrating the location (coordinates) on the filter to which the raw material liquid is applied. Is.
The number of coordinates (locations on the filter) in the plan view of the filter is not particularly limited, but the lower limit of the number of coordinates is one or more, preferably two or more, or three or more. .. The upper limit of the number of coordinates is not particularly limited, but is, for example, 20 or less, 15 or less, and 10 or less. When the number of distances (Z-axis coordinates) between the filter and the discharging means described later is two or more, even if the number of coordinates (locations on the filter) in the plan view plane of the filter is one. It may be two or more places. On the other hand, when the number of distances (Z-axis coordinates) between the filter and the discharging means described later is one, the number of coordinates (locations on the filter) in the plan view plane of the filter is two or more.

The coordinates may be separated from each other (the intervals may be equal or unequal), and may be adjacent or connected. In the present specification, the fine movement of the discharge opening caused by the unintentional fine movement of the discharge means (the fine movement that cannot be technically braked) is not treated as the difference in coordinates. Specifically, the coordinates adjacent to or connected to each other are not separated from each other, but the coordinates in the plan view of the filter are the area of the orbit to which the discharge opening of the pipette tip 40 (an example of the discharge means) has moved. As long as it is at least twice the discharge opening area of the pipette tip 40, it is considered to be two or more points in the present invention (in other words, the area of the trajectory to which the discharge opening of the pipette tip 40 has moved is 2 of the discharge opening area of the pipette tip 40. If it is less than double, the number of coordinates is considered to be one). Further, the arrangement of the coordinates is not particularly limited, and may be symmetric (point symmetric or line symmetric), asymmetric, aligned or not.

The method of discharging the raw material liquid is not particularly limited, and the discharge rate may be constant or non-constant, and may be intermittent or continuous. In addition, when discharging at two or more coordinates per pipette tip 40, the pipette tip 40 moves between the coordinates, but the discharge speed at that time is when it is located at the coordinates (or when it is stopped). It may be the same or different when it is located between the coordinates (or when it is moved) (it is not necessary to discharge when it is positioned or moved between the coordinates). For example, FIGS. 3 (j) and 3 (k) show a zigzag shape and a spiral shape as the movement trajectory of the pipette tip 40 between the coordinates represented by the solid circles, which are represented by the circles. The discharge rate may be the same or different depending on whether it is located at the coordinates or between the coordinates (it is not necessary to discharge when it is located between the coordinates).

Returning to FIG. 1, the Z-axis drive mechanism 73 is slidably connected to the Y-axis drive mechanism 72 (however, it may be to the X-axis drive mechanism 71), and the pipette tip 40 The distance (Z-axis coordinates) between the tip and the cell filtration filter 11 can be adjusted reproducibly. As a result, the linear velocity of the raw material liquid when applied to the filter 11 can be reproducibly adjusted, and the quality (particularly the survival rate) such as the recovery rate and the survival rate of the target cells in the filtered liquid can be reproducibly improved. .. The tendency is that the linear velocity decreases during the free fall of the raw material liquid into the filter by increasing the distance to some extent. As described above, the Z-axis drive mechanism 73 is also a means for variably regulating the coordinates with respect to the filter, and is also an example of a means (described later) for variably regulating the operating conditions of the discharge speed.

In the present embodiment, since the XY coordinates can be two or more, the number of distances (Z-axis coordinates) between the filter and the discharge means is not particularly limited, and may be one or two or more. It may be.

In one preferred embodiment, such regulatory means is controlled by a computer programmed to move the coordinates relative to the discharging filter by a discharging means such as a pipette tip 40. Also, in another embodiment, such regulatory measures are manually manipulated. For example, in a completely closed system automatic culture device, when the discharge means and the cell filtration filter are incorporated, a robot arm arranged in the closed system so as to move the coordinates of the raw material liquid containing the target cells with respect to the filter. Etc. may be operated by a joystick or the like controlled by the user from a position outside the closed system.

As long as the distance (Z-axis coordinates) between the filter and the discharging means differs by, for example, 2 mm or more (preferably 5 mm or more), it is considered to be two or more in the present invention (in other words, if the difference is less than 2 mm, the Z-axis coordinates). The number is considered to be one). In the present specification, the fine movement of the discharge opening caused by the unintentional fine movement of the discharge means (the fine movement that cannot be technically braked) is not treated as the difference in coordinates. Specifically, when discharging at two or more Z-axis coordinates per pipette tip 40, the pipette tip 40 moves between the coordinates, but the discharge speed at that time is when it is located at the coordinates. It may be the same or different when it is located between the coordinates (or when it is stopped) and when it is located between the coordinates (or when it is moved) (it is not necessary to discharge when it is positioned or moved between the coordinates).

Further, in a preferred embodiment of the cell manufacturing apparatus according to the present invention, a means for variably regulating the operating conditions of the discharge rate may be further provided. The rate of discharge affects the recovery rate of target cells (particularly viable target cells) and the survival rate of target cells. Therefore, by variably regulating the operating conditions of the discharge rate, the quality such as the recovery rate and the survival rate of the target cells (particularly the surviving target cells) can be improved with reproducibility.

As the X-axis drive mechanism 71, the Y-axis drive mechanism 72, and the Z-axis drive mechanism 73, any known mechanism can be used, for example, a stage (for example, x stage, xy stage or xyz stage), a valve ( For example, electromagnetic valves or air valves), gears, motors (eg electric motors or stepping motors), pistons, brakes, cables, ball screw assemblies, rack and pinion mechanisms, grippers, arms, pivot points, joints, translational elements, or It may include one or more elements such as other mechanical or electrical elements. Further, for example, the structure supporting the discharging means may include one or more robot elements. For example, the structure supporting the discharge means may include a robot arm that can be freely displaced in the xy direction or the xy direction.

The mechanism that variably regulates the operating conditions of the discharge speed is not particularly limited to the Z-axis drive mechanism 73, and for example, a pump whose delivery pressure is variable by a control signal sent from a controller controlled by a computer or the like. It may be.

The means for collecting the filtered liquid containing the target cells is not particularly limited as long as it can temporarily accept the filtered liquid, and may be various containers or channels.

Examples of the container body as a means for collecting the filtered liquid containing the target cells include a centrifuge tube, a test tube, a beaker, a flask, a tube, a vial for freezing, a cell culture container (including a multi-well plate and a petri dish), and a nano. Examples include micro / micro porous non-woven fabrics (filter paper), funnels, clock plates, cell counter plates, RNA recovery / purification kits, filtrate recovery spoons, and cell seeding reservoirs. Examples of the material of the container include polystyrene, polypropylene, polyethylene, glass, acrylic, and the like. If it is necessary to observe the cells in the filtrate, polystyrene having excellent transparency is preferable. The shape and size of the container are not particularly limited.

The cell production apparatus according to one embodiment of the present invention may further include means for variably regulating the liquid passage time of the cell filtration filter.
The liquid passing time in the present invention is the time required for the raw material liquid to pass through a certain point on the filtration filter. The liquid passing time can be determined by changing the coordinates of the Z axis, changing the discharge speed, changing the linear velocity, the shape of the discharge means, the hole diameter of the discharge part, the number of discharge means, and the material and structure of the filtration filter. By changing to a filtration filter with different properties, it can be regulated variably. By regulating the liquid passing time, it is possible to improve the survival rate of the cells contained in the filtrate and improve the recovery rate of the cells contained in the filtrate.
The linear velocity in the present invention is the velocity of the content liquid when the raw material liquid passes through a certain point on the filtration filter. The linear velocity may be appropriately set and set according to other conditions and is not limited to a specific numerical range. For example, the lower limit is 50 mm / sec or more, 500 mm / sec or more, 1000 mm / sec. As mentioned above, 3000 mm / sec or more is preferable, and the upper limit is preferably 5000 mm / sec or less.
Further, the discharge rate is not limited to a specific numerical range as long as it is appropriately set and set according to other conditions, but for example, the lower limit is 2 μL / sec or more, 10 μL / sec or more, 100 μL /. It is preferably sec or more, 250 μL / sec or more, and 500 μL / sec or more, and the upper limit is preferably 5000 μL / sec or less, 2000 μL / sec or less, 1000 μL / sec or less, and 850 μL / sec or less. The material of the filtration filter is not particularly limited, and examples thereof include Polytetherafluoroethylene (PTFE), Polyvinylidene Fluoride (PVDF), and cellulose mixed ester.
Examples of the structure of the filtration filter include a filter hole diameter, a pore diameter gradient, and a filter thickness. The filter hole diameter has, for example, 0.1 μm or more, 0.2 μm or more, 0.4 μm or more, and 0. 8 μm or more is preferable.
Further, the filtration filter may be given hydrophilic or hydrophobic properties, for example, by subjecting it to a surface treatment. By applying a surface treatment to the early filtration filter, the wettability and water contact angle of the filter can be adjusted. From the viewpoint of filtering the target cells with a good survival rate and recovery rate, the water contact angle θ is preferably larger than 1 ° and smaller than 80 °. The water contact angle in the present invention refers to the angle at which the liquid surface forms the solid surface at the boundary line where the three phases come into contact when the solid surface is in contact with the liquid and the gas.
Further, the filtration filter may be a single layer or a plurality of layers, and can be appropriately selected depending on the type and amount of the target cell and the raw material liquid.

In the cell manufacturing apparatus according to the embodiment of the present invention, in the operation of applying the raw material liquid containing the target cells to the cell filtration filter, the number of times of the discharge, the number of the discharge means (for example, the pipette tip 40) used, or the discharge is performed. It may have means for variably or non-variably regulating one or more additional operating conditions such as discharge pressure at the time of operation.
The number of discharges determines the amount of the raw material liquid discharged to the cell filtration filter when the maximum amount of the raw material liquid discharged at one time is determined. Therefore, by regulating the number of discharges, the amount of the raw material liquid discharged to the cell filtration filter can be set to be reproducible.
The number of means for discharging is useful when the production time of the target cell product is shortened when the discharge rate cannot be increased due to restrictions such as the type of target cells and desired performance (for example, survival rate). That is, by regulating the number of discharge means used, it is possible to set the discharge amount of the raw material liquid to the cell filtration filter per hour without depending on the discharge rate. Specific examples of the discharging means are as described above.

The discharge pressure can be variably regulated by changing the structure of the discharge means, the surface area of the tip portion, and the pressure of the pump of the device for controlling the discharge, for example, the pipette tip 40.
By regulating the discharge pressure, the discharge speed can also be variably regulated. The discharge pressure may be appropriately set according to the characteristics of the target cells contained in the raw material liquid, the viscosity and composition of the raw material liquid, and is not particularly limited. For example, the lower limit is 0.1 MPa or more, 0. 5 MPa and 1 MPa are preferable, and the upper limit of the discharge pressure is not particularly limited as long as the target cell does not cause cell death, but for example, 10 MPa or less and 5 MPa or less are preferable.
The characteristics of the target cell include, for example, resistance to shear stress.
By regulating the discharge pressure as described above, the survival rate and recovery rate of the target cells can be improved.

In one embodiment of the cell manufacturing apparatus of the present invention, the operating conditions of the coordinates for the filter that discharges may be variable and feedback control may be performed.

The cell manufacturing apparatus according to one embodiment includes means for detecting the number of cells contained in the filtered liquid and means for adjusting the operating conditions based on the detection, in which the operating conditions of the coordinates are variable. The number of cells contained in the filtered liquid is ideally proportional to the amount of the raw material liquid supplied to the cell filtration filter, but when the above-mentioned trap phenomenon occurs, the number of cells in the filtered liquid is compared with the amount of the raw material liquid supplied. It is accompanied by a phenomenon that the number of cells in the cell is difficult to increase. Therefore, the presence or absence and degree of the trap phenomenon can be estimated by detecting the number of cells contained in the filtered liquid, and if the operating conditions are adjusted during operation based on this, the prevention or deterioration of the trap phenomenon can be efficiently prevented. Can be done
Examples of the detection of the number of cells contained in the filtered liquid include a method of irradiating the filtered liquid in the container with measurement light from the inside or the outside of the container 30 and measuring the turbidity with a spectrophotometer.
Further, the operating conditions are preferably adjusted in consideration of not only the number of cells contained in the filtered liquid but also the supply amount of the raw material liquid to the cell filtration filter, but the supply amount of the raw material liquid to the cell filtration filter. May be measured directly with a flow meter or the like, or may be calculated indirectly by measuring the supply time if the supply amount per unit time is constant.

In the cell manufacturing apparatus according to another embodiment, the operating conditions of the coordinates are variable, and the means for detecting the accumulation of the filter matter in the coordinates with respect to the filter performing the discharge or the event correlating with the accumulation, and the operating conditions based on the detection. It is provided with a means for adjusting.
The presence or absence and degree of the trap phenomenon can be grasped by the detection, and if the operating conditions are adjusted during the operation based on the detection, the trap phenomenon can be effectively prevented or deteriorated.
The event that correlates with the accumulation of the filter in coordinates with respect to the filter is not particularly limited, and may be the presence or absence and size of the accumulated filter (reference numeral 5 in FIG. 2), the weight increase of the filter due to the accumulation, and the like. ..
The accumulated filter matter can be detected, for example, by imaging the surface of the filter continuously or at regular intervals and analyzing the image.

FIG. 4 is a diagram showing a flow of cell production according to an embodiment of the present invention. For example, as shown in FIG. 4, the manufacturing apparatus according to one embodiment may include means for attaching the cell filtration filter 11 to the opening of the container 30. By mechanically mounting the filter by the device, not only the discharge and recovery steps, but also the effect that the previous filter mounting may have on the target cells should be eliminated, and the entire process should be performed in a clean environment. Is also possible.

The mounting means corresponds to the means 20 for gripping the support 12 for supporting the two or more cell filtration filters 11 or the container 30 and the means for transporting the gripped support 12 and corresponding to the two or more cell filtration filters 11. It may be provided with means for aligning the positions with the openings of the two or more containers 30.
In order to increase the number of target cells in the filtered liquid while reducing the discharge amount of the raw material liquid per coordinate of the cell filtration filter 11, the number of cell filtering filters 11 is increased and the raw material liquid is applied to each filter 11 in parallel. It is useful to discharge. However, as the number of filters 11 and the containers 30 to which each of the filters 11 is mounted increases, the amount of work and difficulty of mounting (for example, failure to mount the filter 11 horizontally) increases, and the filters are mounted. Can have a large effect on the target cells.
According to the above embodiment, by performing this step mechanically by the apparatus including alignment, it is possible to improve the production efficiency of the target cell product while eliminating the influence that may be given to the target cell.

The manufacturing apparatus according to one embodiment may include means for detaching the cell filtration filter 11 from the opening of the container 30. By mechanically removing the filter by the device, not only the discharge and recovery steps but also the effect that the subsequent removal of the filter can have on the target cells (for example, failure to mix the filter medium on the filter) is eliminated. However, it is also possible to carry out the entire process in a clean environment.

Explaining the flow of cell production with reference to FIG. 4, the cell filtration filter 11 is provided with a filtration membrane at the bottom of a tubular object having a collar. Two or more of the cell filtration filters 11 are inserted into holes substantially matching the cross-sectional shape of the tubular object, and are locked and supported by the support 12 by the collar portion. The support 12 in this state is gripped by the cell filtration filter unit gripping arm 20 which is freely movable in each direction of the XYZ axes, and the upper position of the opening of the container 30 (determined by the X-axis and the Y-axis constituting the horizontal plane). And then to the height of the opening (Z-axis) of the container 30, and the cell filtration filter 11 is fitted into the opening. At this point, if the cell filtration filter 11 is perfectly and accurately aligned with the opening, the cell filtration filter 11 is mounted in the opening in the correct orientation (typically horizontal).

However, as the number of filters 11 to be mounted at the same time increases as in the present embodiment, it becomes difficult to successfully mount all the filters 11 in one alignment. In a typical example of mounting failure, the filter 11 is caught on the edge of the opening of the container 30 during fitting and is mounted in an inclined state. Therefore, it is preferable to move the filter 11 finely in the horizontal direction while the cell filtration filter 11 is fitted to the opening of the container 30. As a result, the filter 11 that is not mounted in the correct posture is also correctly aligned while being finely moved, and the possibility that the filter 11 is mounted in the correct posture is increased.

In this way, the cell filtration filter 11 and the opening of the container 30 are aligned, and the cell filtration filter 11 is attached to the opening (ab). Since the alignment means itself is well known, the description thereof will be omitted.

Next, the raw material liquid is discharged from the pipette tip 40 to each cell filtration filter 11 to perform filtration (c). In FIG. 4C, there is one pipette tip 40 that discharges to the plurality of filters 11, but the number of pipette tips 40 can be appropriately set as described above.

After collecting the filtered liquid into each container 30, the cell filtration filter unit gripping arm 20 is operated in the reverse procedure to the mounting operation described above to separate the cell filtration filter 11 from the opening of the container 30.

Although the support 12 in FIG. 4 supports two or more filters 11, two or more containers 30 may be supported.

The cell production apparatus according to the embodiment of the present invention may further include means for recovering target cells by solid-liquid separation from the collected filtered liquid. By mechanically performing the recovery process of the target cells by the apparatus, it is possible to eliminate the influence that the recovery step may have on the target cells and to perform all the steps in a clean environment.

The means for recovering the target cells by solid-liquid separation is not particularly limited, and examples thereof include centrifugation and sorting with hollow threads. Of these, centrifugation is preferred because it is simple and efficient. The recovery means includes a means for closing the opening of the container and a means for applying a centrifugal force to the container with the closed opening so that the filtered liquid and the target cells do not flow out from the container in the process of centrifugation. Is preferable.

FIG. 5 shows the flow of cell production according to the embodiment of the present invention. A gripping arm 22 similar to that shown in FIG. 4 moves the lid 50 from above the opening to bring it into close contact with the container 30 containing the filtrate containing the target cells 1, and closes the opening (a to b). ).

The lid 50 in FIG. 5 is a flat plate and has a sufficient area to cover the openings of a plurality of containers 30. From the viewpoint of closing the opening with high accuracy, it is preferable that the lid 50 is provided with a recess that substantially matches the outer shape of the opening, and the opening of the container 30 is fitted into the recess.

Next, the target cells 1 are separated from the liquid component of the filtered liquid by means 60 for applying centrifugal force to the container 30 (b). If necessary, the washing liquid may be added to the target cells of the container 30 to disperse the cells, and the washing liquid may be removed by centrifugation again.

Next, a pharmaceutically acceptable medium or medium is added to the container 30 to suspend the target cells 1 to prepare a product (c), and then the lid 50 is removed to open the opening of the container 30 (). d). The removal operation of the lid 50 may be the reverse procedure of the attachment operation.
In the present invention, the pharmaceutically acceptable medium is not particularly limited as long as it is a solution used in human treatment, and for example, physiological saline solution, 5% dextrose solution, Ringer's solution, Ringer's lactate, Ringer's acetate solution, and the like. Examples include solution (No. 1 solution), dehydration replenishment solution (No. 2 solution), maintenance infusion solution (No. 3 solution), and postoperative recovery solution (No. 4 solution).
Further, for example, DMSO, glycerol, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran trehalose, HES and the like can be added to the container 30 to suspend the target cells 1 to prepare a product. ..

The apparatus according to one embodiment further refers to a database that stores data relating to the correspondence between the coordinates of the discharging filter and the recovery rate of target cells in the product, and the required database. Further means may be provided for selecting the operating conditions of the coordinates for achieving the recovery rate and for the discharging filter. By discharging by utilizing the stored correspondence data, the target cells can be recovered with a higher expected value and a required recovery rate.

The apparatus according to one embodiment further stores data relating to the correspondence between the discharge rate and the survival rate of target cells in the product, and the means of selection is the required survival rate. The operating conditions of the discharge rate for achieving the above may be selected. By discharging using the stored correspondence data, it is possible to recover the target cells having the required survival rate with a higher expected value.

In the present invention, the target cell is not particularly limited, and has properties (size and filter affinity) that can be separated from other components (cells and cell masses other than the target cell, cultured debris (cell debris), contaminants, etc.) by filter filtration. ) And may be any cell that requires isolation. Although cells derived from all living organisms such as animals, plants, and insects or artificial cells can be mentioned, cells derived from animals, particularly cells derived from humans, are preferable because of the great need for the effects of the present invention.

For example, cells collected from animal body fluids and tissues, artificially cancerous strained cells, and those obtained by culturing these cells in vitro are mentioned, and preferably collected from human body fluids and tissues. Cells or cultured cells, such as human bone marrow fluid, blood (including peripheral blood, G-CSF mobilized peripheral blood, etc.), umbilical cord blood, menstrual blood, adipose tissue, liver tissue, pancreatic tissue, heart tissue, Includes cells collected from nerve tissue, tumor tissue, etc. or cultured cells.

The colony forming cells are not particularly limited as long as they form colonies when the cells are cultured in vitro under arbitrary conditions, and examples thereof include progenitor cells and stem cells. Examples of progenitor cells include progenitor cells such as vascular endothelial progenitor cells, neural progenitor cells, and hepatic progenitor cells. Examples of stem cells include hematopoietic stem cells, mesenchymal stem cells or adipose-derived stem cells, somatic stem cells such as cancer stem cells, and pluripotent stem cells such as ES cells or iPS cells.

Adhesive colony-forming cells refer to cells that adhere to and proliferate on arbitrary scaffolds when the cells proliferate, such as vascular endothelial progenitor cells, neural progenitor cells, and hepatic progenitor cells. Examples thereof include progenitor cells, somatic stem cells such as mesenchymal stem cells and adipose-derived stem cells, and pluripotent stem cells such as ES cells and iPS cells, but the present invention is not particularly limited.

Non-adhesive (floating) colony-forming cells refer to cells that can proliferate without adhering to a scaffold when the cells proliferate, such as somatic stem cells such as cancer stem cells and neural stem cells. Examples thereof include pluripotent stem cells such as ES cells and iPS cells.

Further, the target cell in the present invention may be a differentiated cell obtained by inducing differentiation of a pluripotent stem cell. Examples of the differentiated cells include endoderm cells, mesoderm cells, ectoderm cells, and somatic cells.

Endodermal cells are tissues of organs such as the digestive tract, lungs, thyroid, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, thoracic membrane, laryngeal, ear canal, trachea, bronchi, and urethra (bladder, It has the ability to differentiate into most of the urethra, part of the urethra, etc., and is commonly referred to as the germ layer (DE). Differentiation from pluripotent stem cells to endoderm cells can be confirmed by measuring the expression level of genes specific to endoderm cells. Examples of genes specific to endoderm cells include SOX17, FOXA2, CXCR4, AFP, GATA4, EOMES and the like.

Mesothelial cells include the mesothelium and the mesothelium, muscles, skeleton, cutaneous dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymph vessels, spleen, kidneys, which support the body cavity. It differentiates into the ureter and gonads (testis, uterus, gonad epithelium). Examples of genes specific to mesoderm cells include MESS1, MESS2, FOXF1, BRACHYURY, HAND1, EVX1, IRX3, CDX2, TBX6, MIXL1, ISL1, NSAI2, FOXC1 and PDGFRα.

Ectoderm cells include the epidermis of the skin and the epithelium of the terminal urinary tract of men, hair, nails, skin glands (including mammary glands and sweat glands), and sensory organs (including the epithelium of the oral cavity, pharynx, nose, and terminal end of the rectum. (Salivary gland) Form the crystalline lens. Some ectoderm cells invade into grooves during development to form neural tubes, and are also the source of neurons and melanocytes in the central nervous system such as the brain and spinal cord. It also forms the peripheral nervous system. Examples of genes specific to ectoderm cells include FGF5, OTX2, SOX1, PAX6 and the like.

The somatic cells in the present invention are not particularly limited as long as they can induce differentiation from pluripotent stem cells and can exist in vivo, but for example, somatic stem cells (bone marrow, adipose tissue, dental pulp, placenta, etc.) Membranous stem cells, neural stem cells, etc. derived from egg membrane, umbilical cord blood, sheep membrane, chorionic villi, etc.), nerve cells, glial cells, oligodendrocytes, Schwan cells, myocardial cells, myocardial progenitor cells, hepatocytes, liver progenitor cells , Α cells, β cells, fibroblasts, cartilage cells, corneal cells, vascular endothelial cells, vascular endothelial precursor cells, peri-cells, skeletal muscle cells, giant nuclei, hematopoietic stem cells, airway epithelial cells, germ cells, dendritic cells, Examples thereof include eosinophils, obese cells, T cells, erythropoietin-producing cells, intestinal epithelium, alveolar epithelial cells, kidney cells, etc., and the cells are in a gene-introduced form or a form in which a target gene on the genome is knocked down. It may be.

Cancer stem cells, like stem cells, are pluripotent and have the ability to proliferate indefinitely, and are resistant to therapeutic agents.

The liquid contained in the raw material liquid containing the target cells is not particularly limited as long as it is a liquid exhibiting fluidity, and examples thereof include liquids such as physiological saline, buffer solution, medium, and washing solution.

As the medium, a basal medium may be used, or an additive may be added to the basal medium. As the basal medium, for example, Neurobasal medium, NeuroProgenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium EM medium, Medium , ΑMEM medium, DMEM medium, DMEM / F12 medium, ham medium, RPMI 1640 medium, Fisher's medium, and a mixed medium thereof, which are not particularly limited as long as they can be used for culturing animal cells. When culturing iN or iMN, a mixture of Neurobasal medium and DMEM / F12 is preferably used.

Additives are not particularly limited, but are serum, retinoic acid, Wnt, BMP, bFGF, EGF, HGF, Sonic hedgehog (Sh), neurotrophic factor family, insulin-like growth factor 1 (IGF1), amino acids, vitamins, inter. Examples thereof include substances necessary for cell proliferation or survival such as leukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, and antibiotics. When culturing iN or iMN, retinoic acid, Sh, BDNF, GDNF, NT-3, B27-supplement and N2-supplement are preferably used.
These additives may be added all at once, or may be changed stepwise according to the manufacturing process of the product containing the target cells.

The washing solution is not particularly limited as long as it can wash away cells, cell clumps, cultured debris (cell debris) and impurities to be removed, but for example, physiological saline, Ringer's solution, medium used for cell culture, phosphate buffer, etc. Examples include a general buffer solution of the above, or a solution obtained by adding serum or protein to these solutions.

(Manufacturing method of cell products)
The method for producing a cell product according to the present invention includes a step of producing a cell product using the cell production apparatus as described above. The quality of the target cells to be recovered and the recovery rate thereof can be improved.

The method for producing a cell product may include a step of isolating cells from a tissue. In addition, the method for producing a cell product may include a step of culturing cells. Further, the method for producing a cell product may include a step of mixing cultured cells with a pharmaceutically acceptable medium or medium to formulate the cells. In addition, the method for producing a cell product may include a step of cryopreserving cultured cells or a formulated cell preparation.
As the culturing method in the culturing step, suitable culturing conditions may be appropriately selected according to the type of cells, a conventional culturing method can be used, or new culturing conditions can be set.
The cell is not particularly limited as long as it can produce the target cell contained in the cell product, and examples thereof include the above-mentioned cell.

The steps that require filtration with a cell filtration filter are not particularly limited, but for example, a step of isolating cells from a tissue, a step of culturing cells, a formulation step, a cryopreservation step, a commercialization step, and a characteristic analysis of target cells. Can be mentioned. Examples of the characteristic analysis of the target cell include gene analysis, RNA extraction, chromosome analysis, FACS, and intracellular metabolism analysis.

(server)
The server according to the present invention refers to a database that stores data relating to the correspondence between the coordinates of the filter for discharging and the recovery rate of target cells in the product, and the database to obtain the required recovery rate. A means for selecting an operating condition of coordinates with respect to the filter for discharging to achieve the above, and a means for transmitting information on the selected operating condition to an apparatus for manufacturing a product containing the target cell described above. By discharging by utilizing the stored correspondence data, the target cells can be recovered with a higher expected value and a required recovery rate. Further, by centrally managing data on a server physically separated from the cell manufacturing apparatus serving as a terminal, the terminal of the cell manufacturing apparatus can be simplified.

In one embodiment of the server according to the invention, the database further stores data relating to the correspondence between the discharge rate and the viability of the target cell in the product, and the means of selection is the required said. The means for selecting and transmitting the operating conditions of the discharge rate for achieving the survival rate may transmit the information to a cell manufacturing apparatus that variably regulates the discharge rate. By discharging using the stored correspondence data, it is possible to recover the target cells having the required survival rate with a higher expected value. Further, by centrally managing data on a server physically separated from the cell manufacturing apparatus serving as a terminal, the terminal of the cell manufacturing apparatus can be simplified.

It is preferable that the data in the database is classified according to the type of target cell and the type of filter. As a result, the required quality such as recovery rate and survival rate can be realized with higher expected values.

The server hardware is not particularly limited, and a conventionally known general-purpose server device can be used.

(system)
The cell manufacturing system according to the present invention includes an apparatus for manufacturing a product containing the above-mentioned target cells and the above-mentioned server.

In the system according to one embodiment, the apparatus manufactures the product based on the operating conditions of the information transmitted from the server, and as a result, the discharge rate and the viability of the target cells in the product. The actual data regarding the correspondence relationship between the above or the coordinates with respect to the filter performing the discharge and the correspondence relationship between the recovery rate of the target cells in the product is transmitted to the server, and the database additionally stores the actual data. Has a configuration. As the amount and type of data stored in the database increases, the accuracy of the operating conditions of coordinates or discharge rates recommended based on the data can continue to improve.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to such Examples. The evaluation of the characteristic values shown in the examples was measured by the following methods.

Example 1
In the cell filtration step, it was confirmed whether or not the cell recovery amount was affected by the coordinates with respect to the filter when the raw material liquid containing the iPS cells (target cells) was discharged to the cell filtration filter. In this embodiment, the coordinates of the filter in the plan view were changed, and the distance between the ejection means and the filter was fixed.

The experiment was carried out according to the following procedure.
1. 1. The frozen solution of iPS cells (RPC-SF-iM strain) was thawed in a constant temperature bath at 37 ° C.
2. 2. The thawed solution was diluted in 5 mL StemFit medium and centrifuged at 1500 rpm for 3 min.
3. 3. Following removal of culture supernatant, the iPS cells cell suspension (raw material liquid) was prepared in 950uL at a concentration of 1.44x10 ⁵ cells / mL, on a cell strainer filter than the discharge means (pipette tip 40) Discharged. The discharge rate of the cell suspension was set to either 100 μL / sec, 500 μL / sec, or 900 μL / sec.
The method of discharging the raw material liquid is as follows: (1) the total amount to one coordinate on the filter (cell strainer 100 μm mesh) as shown in FIG. 3 (b), and (2) on the filter as shown in FIG. 3 (c). Half amount to 2 coordinates (repeat twice), (3) 1/4 amount to 4 coordinates on the filter as shown in Fig. 3 (g) (however, 2 pipette tips 40 are used together and 2 coordinates The discharge is repeated twice at the same time).
4. Measure the number of cells and cell viability contained in the filtered liquid.
5. Based on the formula below, the ratio (%) of the number of cells captured by the filter (the number of uncollected target cells) to the number of uncollected cells was calculated.
(Number of uncollected cells) = (Total number of cells before filtration)-(Total number of cells after filtration)
(Percentage of uncollected cells) (%) = (Number of uncollected cells) / (Total number of cells before filtration) x 100
The results obtained are shown in Table 1.

As shown in Table 1, the number and proportion of uncollected cells and the recovery rate of target cells changed significantly depending on the coordinates of the filter that discharges the raw material liquid (coordinates of the filter in the plan view). Specifically, it was found that by increasing the number of coordinates and reducing the discharge amount of the raw material liquid for each coordinate, the target cells captured on the filter decrease and the target cells contained in the filtered liquid increase. That is, it was found that the recovery rate of the target cells can be reproducibly improved by variably regulating the operating conditions of the coordinates.

Example 2
In the cell filtration step, it was confirmed whether or not the height at which the raw material liquid was discharged (the distance from the discharge port of the discharge means to the surface of the cell filtration filter) affected the target cells to be recovered. In this embodiment, the coordinates of the filter in the plan view were fixed, and the distance between the ejection means and the filter was changed.

The experiment was carried out according to the following procedure.
That is, the raw material liquid ^(1.44x10 5 cells / mL) was prepared in the same manner as steps 1-3 of Example 1, the discharge means the same as that used from (pipette tip 40) in cell strainer filter (Example 1 ) Discharged on.
The discharge rate of the cell suspension was 900 μL / sec.
The height of the discharge port of the discharge means from the filter surface was set to 0 mm under condition 1, 20 mm under condition 2, and 100 mm under condition 3, and the raw material liquid was dropped to one place on the filter. The survival rate of the target cells was calculated based on the following formula.
(Cell viability in filtered liquid) (%) = (Number of viable cells in filtered liquid) / (Total number of cells in filtered liquid) x 100
The results obtained are shown in Table 2.

As shown in Table 2, the total number of cells (recovery rate) in the filtered liquid, the survival rate of the target cells, and the number of the target cells that survive vary greatly depending on the distance from the discharge port of the discharge means to the surface of the cell filtration filter. It was. From this, it was found that the recovery rate and the survival rate of the target cells can be reproducibly improved by variably regulating the operating conditions of the coordinates (distance between the filter and the discharge position) with respect to the filter. It is presumed that the distance affects the linear velocity of the raw material liquid with respect to the filter, and as a result, affects the recovery rate and the survival rate of the target cells.

Example 3
In the cell filtration step, it was confirmed whether the discharge rate of the cell suspension affects the viability of the recovered cells.
The experiment was carried out according to the following procedure.
1. 1. That is, the raw material liquid (1.40 x 10 ⁵ cells / mL) was prepared in the same manner as in steps 1 to 3 in Example 1, and 10 μL / sec, 100 μL / sec, 500 μL / sec, from the discharging means (pipette tip 40). The cells were discharged onto a cell filtration filter (same as that used in Example 1) at a discharge rate of 900 μL / sec and 2000 μL / sec.
2. 2. After collecting the filtered liquid, the total number of cells and the number of viable cells in the filtered liquid were measured, and the survival rate was calculated.

As shown in Table 3, the total number of cells contained in the filtrate was substantially constant regardless of the discharge rate. On the other hand, the cell viability was extremely high when the discharge rate was 500 μL / sec or less, but decreased when the discharge rate was 900 μL / sec or more. From this, it was found that the viability of the target cells and the yield of the surviving target cells can be reproducibly improved by variably regulating the discharge rate of the raw material liquid.

10 Cell Filtration Filter Unit 11 Cell Filtration Filter 12 Support 20 Cell Filtration Filter Unit Grip Arm 30 Container 40 Pipette Tip

Claims (18)

1. A device for manufacturing products containing target cells.
   A means for discharging the raw material liquid containing the target cells to the cell filtration filter,
   A means for collecting the filtered liquid containing the target cells obtained by the cell filtration filter, and
   A means for variably regulating the operating conditions of the coordinates for the filter that discharges,
   A device equipped with.
2. A means for variably regulating the operating conditions of the discharge speed, and
   The device according to claim 1, further comprising.
3. A means for variably regulating the liquid passing time of the cell filtration filter and
   The device according to claim 1 or 2, further comprising.
4. A means for variably or non-variably regulating one or more additional operating conditions selected from the group consisting of the number of discharges, the number of discharge means used, and the discharge pressure.
   The device according to any one of claims 1 to 3, further comprising.
5. The operating conditions are variable
   The device according to any one of claims 1 to 4, further comprising a means for detecting the number of cells contained in the filtered liquid and a means for adjusting the operating conditions based on the detection.
6. The operating conditions are variable
   Any of claims 1 to 5, wherein the device further comprises means for detecting the accumulation of filters or an event correlating with them in coordinates with respect to the filter performing the discharge, and means for adjusting the operating conditions based on the detection. The device described.
7. The device according to any one of claims 1 to 6, further comprising a means for attaching the cell filtration filter to the opening of a container for collecting the filtered liquid.
8. The mounting means include means for gripping two or more of the cell filtration filters or a support supporting the container.
   The device according to claim 7, further comprising means for transporting the gripped support and aligning the two or more cell filtration filters with the opening of the container.
9. The device according to any one of claims 1 to 8, further comprising a means for separating the cell filtration filter from the opening of the container for collecting the filtered liquid.
10. The device according to any one of claims 1 to 9, further comprising a means for recovering target cells by solid-liquid separation from the filtered liquid.
11. The solid-liquid separation is centrifugation,
    The device according to claim 10, wherein the collecting means includes a means for closing the opening of the container in which the filtered liquid is collected and a means for applying a centrifugal force to the container.
12. A method for manufacturing the product using the device according to any one of claims 1 to 11.
13. A database that stores data on the correspondence between the coordinates of the discharging filter and the recovery rate of target cells in the product.
    With reference to the database, means for selecting the operating conditions of the coordinates for the filter performing the discharge in order to achieve the required recovery rate, and
    A server including means for transmitting information on the selected operating conditions to the apparatus according to any one of claims 1 to 11.
14. The database further stores data on the correspondence between the rate of discharge and the viability of the target cells in the product.
    The selecting means selects the operating conditions of the discharge rate to achieve the required survival rate.
    The server according to claim 13, wherein the transmitting means transmits the information to the apparatus according to claim 2.
15. A system including the device according to any one of claims 1 to 10 and the server according to claim 13 or 14.
16. The device manufactures the product based on the operating conditions of the information transmitted from the server.
    As a result, actual data on the correspondence between the discharge rate and the survival rate of the target cells in the product, or the correspondence between the coordinates for the filter performing the discharge and the recovery rate of the target cells in the product. To the server
    The system according to claim 15, wherein the database additionally stores the actual data.
17. A database that stores data on the correspondence between the coordinates of the discharging filter and the recovery rate of target cells in the product.
    The apparatus according to any one of claims 1 to 11, further comprising means for selecting the operating conditions of the coordinates for achieving the required recovery rate and for the discharging filter with reference to the database.
18. The database further stores data on the correspondence between the rate of discharge and the viability of the target cells in the product.
    17. The apparatus of claim 17, wherein the means of selection is the operating conditions of the discharge rate to achieve the required survival rate.

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