Cell selection method, apparatus, and apparatus system

Abstract

The present invention addresses the problem of providing a cell selection method that enables the assessment of the damage tolerance of cells in a floating culture system, a device for selecting cells and a device system for selecting cells. According to the present invention, provided is a cell selection method that comprises introducing cells into a microchannel, passing the cells through the microchannel, assessing the damage tolerance of the cells against a damage caused by the passage of the cells through the microchannel, and selecting cells on the basis of the assessment of the damage tolerance.

Description

Technical Field

[0001] The present invention relates to a method for selecting cells suitable for a mass production process, an apparatus for selecting cells, and an apparatus system for selecting cells.

Background Art

[0002] Conventionally, in the field of antibody drugs, when selecting clones suitable for a mass production process, clones have been selected using the amount of antibody produced by cells as an index. On the other hand, in recent years, in order to increase the productivity of antibodies, a perfusion culture process in which cells are cultured at a high cell density for a long period has attracted attention. Perfusion culture at a high cell density is a culture method that requires high stirring and a high oxygen supply amount, and the damage that cells receive from the fluid in the culture tank is greater than that of conventional fed-batch. Therefore, when clones selected using the amount of antibody produced by cells as an index as conventionally performed are applied to perfusion culture, cell death may occur due to damage from the fluid.

[0003] Due to the above circumstances, in the selection of cells suitable for perfusion culture, it is desirable to pre-select clones with high damage resistance. For example, in Patent Document 1, a fluid is flowed through a chamber in which cells are fixed to apply a shear stress, and the response is evaluated to determine the stirring conditions. In Non-Patent Document 1, a cell suspension is flowed through a microchannel to apply a shear stress, and cell damage in a centrifugation step is evaluated.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In Patent Document 1, cells are in a suspended state during culture, but the evaluation is carried out in a state where the cells are adhered. Further, in Non-Patent Document 1, it is a technique regarding the examination of centrifugation conditions and not a technique regarding cell selection.

[0007] An object to be solved by the present invention is to provide a method for selecting cells that enables the evaluation of cell damage resistance in a suspension culture system. Further, an object to be solved by the present invention is to provide an apparatus for selecting cells and an apparatus system for selecting cells for carrying out the above-described method for selecting cells.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the present inventor has succeeded in evaluating the damage resistance of cells in a suspension culture system by introducing cells into a microchannel, passing them through the microchannel, and evaluating the damage resistance of the cells against the damage received when the cells pass through the microchannel. The present invention has been completed based on the above findings.

[0009] That is, according to the present invention, the following inventions are provided. <1> Introducing cells into a microchannel and passing them through the microchannel, evaluating the damage resistance of the cells against the damage received when the cells pass through the microchannel, and selecting cells based on the evaluation of the above damage resistance, A method for selecting cells, comprising the above steps. <2> The method according to <1>, wherein the damage received by the cells is the stress received from the fluid. <3> The method according to <1> or <2>, wherein the damage resistance of the cells is evaluated based on the damaged state of the cells. <4> Damage resistance is evaluated based on the cell survival rate relative to the energy dissipation rate. <1> from <3> The method described in any one of the following. <5> Damage resistance is assessed based on the rate of lactate dehydrogenase release from cells relative to the rate of energy dissipation. <1> from <3> The method described in any one of the following. <6> Damage resistance is evaluated based on cell viability against shear stress. <1> from <3> The method described in any one of the following. <7> Damage resistance is evaluated based on the rate of lactate dehydrogenase release from cells in response to shear stress. <1> from <3> The method described in any one of the following. <8> By using multiple microchannels and exchanging them, we evaluate the cell's resistance to damage. <1> from <7> The method described in any one of the following. <9> The internal diameter tolerance in multiple microchannels is less than ±10%. <8> Methods used. <10> The microfluidic channels are electroformed pipes. <1> from <9> The method described in any one of the following. <11> The inner diameter of the microchannel is 10 to 3000 μm. <1> from <10> The method described in any one of the following. <12> Microchannels are grooves. <1> from <7> The method described in any one of the following. <13> The groove consists of a combination of a metal plate and a grooved metal plate. <12> Methods used. <14> The width of each side of the groove's cross-section is 10 to 3000 μm. <12> or <13> Methods used. <15> The cells are either Chinese hamster ovary-derived cells or human fetal kidney cells 293. <1> from <14> The method described in any one of the following. <16> The cells that are selected are the cells that produce proteins. <1> from <15> The method described in any one of the following. <17> These are cells that have been multiplied from monoclonal cells. <1> ~ <16> The method described in any one of the following. <18> Cells that produce proteins are cells that produce proteins through perfusion culture. <16> or <17> Methods used. <19> Microchannels and A pressure gauge for measuring the pressure upstream of a microchannel, A first pump for delivering the cell suspension is installed upstream of the microchannel, A second pump for supplying cleaning fluid is installed upstream of the microchannel, A piping for supplying the cell suspension to the first pump, and a piping for supplying the washing solution to the second pump, Piping for supplying the liquid from the first pump and the liquid from the second pump to the microchannel, Piping for collecting the liquid discharged from the above microchannels. A device for selecting cells, including [the specified element]. <20> <19> A cell selection apparatus system comprising a device for selecting cells as described above, and a plurality of microchannels for interchangeable use. <21> The internal diameter tolerance in multiple microchannels is less than ±10%. <20> The apparatus system described above. [Effects of the Invention]

[0010] According to the cell selection method, cell selection apparatus, and cell selection apparatus system of the present invention, damage resistance between cells in a suspension system can be quantified. Furthermore, the damage resistance index can be used to predict the viability and proliferation rate of cells in culture, and can be used as an indicator for cell selection. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows an example of the apparatus of the present invention. [Figure 2] Figure 2 shows an example of a method for fabricating a divided channel by combining a metal plate and a grooved metal plate. [Figure 3] Figure 3 shows the results of measuring the variation in pipe diameter for SUS (Steel Use Stainless) piping and electroformed piping. [Figure 4]Figure 4 shows the results of measuring the repeatability of SUS piping and electroformed piping. [Figure 5] Figure 5 shows the results of two quantitative assessments of damage resistance for three types of clones. [Figure 6] Figure 6 shows the cell culture apparatus used in perfusion culture. [Figure 7] Figure 7 shows the results of measuring cell viability in perfusion culture. [Figure 8] Figure 8 shows the measurement results of the bleeding rate in perfusion culture. [Figure 9] Figure 9 shows the results of quantitative evaluation of damage resistance in 293 human fetal kidney cells. [Modes for carrying out the invention]

[0012] The details of the present invention will be described in detail below. <Method for selecting cells> The cell selection method according to the present invention is Introducing cells into a microchannel and passing them through the microchannel, To evaluate the cell's resistance to damage caused by passing through microchannels, and Select cells based on the above damage resistance evaluation. This method includes [something].

[0013] In the present invention, when evaluating the damage resistance of cells, it is preferable to quantify the damage resistance using the physical properties of the cell culture medium. Preferably, the damage resistance of cells can be evaluated based on the state of cell damage.

[0014] As an example, it is possible to compare the cell density before damage and the cell density after damage. That is, it is possible to compare the cell density before passing through the microchannel and the cell density after passing through the microchannel. For the cells before damage and the cells after damage, the damage resistance of the cells can be evaluated using the amount of substance of the components discharged from the cells. Particularly preferably, the damage resistance of the cells can be evaluated using, as indices, the dead cell density, the live cell density, the cell viability, the LDH (lactate dehydrogenase) concentration, the HCP (host cell-derived protein) concentration, the DNA concentration, the turbidity, and the like.

[0015] In the present invention, the Reynolds number (Re) [-], the density (ρ) [kg / m 3 , the flow velocity (u) [m / s], the inner diameter of the pipe (d) [m], the viscosity (μ) [Pa·s], the flow rate (U) [m 3 / s], the energy dissipation rate (EDR) [W / m 3 , the pressure loss (ΔP) [Pa], the pipe length (l) [m], and the loading time (t) [s] have the following relationships. Re = (ρ × u × d) / μ u = 4U / (πd 2 ) EDR = (d 2 × ΔP 2 ) / (16 × l 2 × μ) t = (π × d 2 × l) / (4U)

[0016] In the present invention, the energy dissipation rate (EDR) is preferably 10 to 10 11 [W / m 3 , more preferably 10 5 ~10 11 [W / m 3 , particularly preferably 10 5 ~10 10 [W / m 3 ; and most preferably 2.0 × 10 9 ~3.0 × 10 9 [W / m 3The energy dissipation rate can be used as an indicator of damage to cells. In this specification, unless otherwise specified, the notation "A~B" indicating a numerical range means a numerical range that includes the lower and upper limits. Let Re be in the laminar flow region, where Re ≤ 2000. The time during which damage is applied (load time (t)) is preferably 1.0 × 10⁻⁶ -7 The interval is between 100 seconds and 1.0 × 10⁻⁶ seconds, preferably 1.0 × 10⁻⁶. -6 It is between 50 seconds and more preferably 1.0 × 10 -5 It is between 10 seconds. This disclosure specifies that the energy dissipation rate is preferably 10 to 10 11 [W / m 3 ] and more preferably 10 5 ~10 11 [W / m 3 ], particularly preferably 10 5 ~10 10 [W / m 3 ], and most preferably 2.0 × 10 9 ~3.0×10 9 [W / m 3 It can be used to select cell clones for use in the culture method described above.

[0017] In the present invention, damage resistance can preferably be evaluated based on the cell viability rate relative to the energy dissipation rate. The energy dissipation rate is an indicator of the magnitude of cell damage caused by the fluid. The greater the energy dissipation rate, the greater the cell damage and the lower the cell viability rate. By passing cells through a microchannel, the cells pass through a space with a certain energy dissipation rate, causing damage to the cells. The cell viability rate relative to the energy dissipation rate refers to the cell viability rate after the cells have passed through a space with a certain energy dissipation rate. The energy dissipation rate when passing through a microchannel can be determined by measuring the pipe diameter, viscosity, pipe length, and pressure drop. Cell viability can be measured by known methods, but it is preferable to measure it using Vi Cell XR. In the present invention, damage resistance can preferably be evaluated based on the rate of lactate dehydrogenase release from cells relative to the energy dissipation rate. The rate of lactate dehydrogenase release relative to the energy dissipation rate refers to the rate of lactate dehydrogenase release after a cell has passed through a space having a certain energy dissipation rate.

[0018] Preferably, the damage the cells receive is due to stress from the fluid. The motion of cells in a fluid can be broken down into four basic elements: translation, stretching, shear deformation, and rotation. Of these, stretching and shear deformation are the motions that deform cells. Of the expansion and contraction, elongation is due to the flow velocity u in the flow axis x direction. x However, this is a deformation when increasing in the x direction (δu x (δx > 0), compression is due to the flow velocity u in the x-direction of the flow axis. x However, this is the deformation when it decreases in the x direction (δu x (δx < 0). Shear deformation is caused by the flow velocity u in the flow axis x direction. x However, this is the deformation when it changes in the y direction (δu x ( / δy≠0). Under the complex fluid dynamics of a culture vessel, cells are subjected to elongation, contraction, and shear stress.

[0019] As mentioned above, the types of damage include expansion and contraction and shear stress. Preferably, the damage is due to shear stress.

[0020] In the present invention, damage resistance can preferably be evaluated based on the cell viability against shear stress. Shear stress is an indicator of the magnitude of cell damage caused by fluid, and the higher the shear stress, the lower the cell viability. Shear stress can be calculated from the energy dissipation rate and the viscosity of the cell suspension. Cell viability relative to shear stress refers to the cell survival rate after a cell has passed through a space with a certain shear stress. The shear stress when passing through a microchannel can be determined by measuring the pipe diameter, viscosity, pipe length, and pressure drop. While cell viability can be measured by known methods, it is preferable to measure it using Vi Cell XR. Shear stress (τ) is determined by viscosity (μ) and flow velocity u in the x-direction of the flow axis. x Change in the y direction (δu x The following relationship exists between / δy) and the above. Shear stress (τ) [Pa] = μ × (δu x / δy) In the present invention, the shear stress (τ) is preferably 1.0 × 10⁻⁶. ―1 ~1.2 × 10 4 [Pa], more preferably 1.0 × 10 1 ~1.2 × 10 4 [Pa], particularly preferably 1.0 × 10⁻⁶ of the lethal region of CHO cells 1 ~3.6×10 3 [Pa]. Most preferably 1.5 × 10 3 ~1.9×10 3 It is [Pa]. In the present invention, damage resistance can preferably be evaluated based on the rate of lactate dehydrogenase release from cells in response to shear stress.

[0021] The morphology of the microchannels is not particularly limited, but for example, pipes with small inner diameter tolerances made by electroforming (electroformed pipes), or segmented channels made by combining a metal plate and a grooved metal plate in a way that allows for disassembly and cleaning can be used. In the case of segmented channels, the microchannels are grooves.

[0022] When the microfluidic channel is an electroformed pipe, the inner diameter of the electroformed pipe is generally 10 μm to 3000 μm, preferably 20 μm to 2000 μm, more preferably 30 μm to 1000 μm, even more preferably 40 μm to 500 μm, and particularly preferably 50 μm to 300 μm. When the microchannel is an electroformed pipe, the cross-section of the channel is preferably circular. Also, in this specification, electroformed pipe and electroformed piping mean the same thing.

[0023] When the microchannel is a groove, the width of each side of the groove's cross-section is generally 10 μm to 3000 μm, preferably 20 to 2000 μm, more preferably 30 to 1000 μm, even more preferably 40 to 500 μm, and particularly preferably 50 μm to 300 μm.

[0024] The length of the microchannel is generally 10 mm to 2000 mm, preferably 20 mm to 500 mm, more preferably 30 mm to 100 mm, and particularly preferably 40 mm to 60 mm.

[0025] In this invention, multiple microchannels can be used, and the damage resistance of cells can be evaluated by exchanging the microchannels. When using multiple microchannels as described above, the internal diameter tolerance of the multiple microchannels is preferably less than ±10%, more preferably within ±8%, even more preferably within ±5%, and particularly preferably within ±3%. Lowering the internal diameter tolerance can improve the reproducibility of damage resistance evaluation. When evaluating multiple clones in antibody production, it is preferable to have multiple microchannels because clogging can occur in a single microchannel. The number of clones to be evaluated may be 3 or more, 10 or more, or 100 or more. Furthermore, the number of clones selected that have high damage resistance may be 3 or less, 5 or less, 10 or less, or 50 or less.

[0026] The type of cell used in this invention is not particularly limited, but is preferably an animal cell, and more preferably a mammalian cell. The cell may be a primary cell or a cell line. The cell may also be a genetically modified cell (for example, a cell into which a gene has been introduced from an external source).

[0027] Examples of cells include, but are not limited to, animal cells such as Chinese hamster ovary cells (CHO cells), human embryonic kidney cells 293 (also known as HEK293), monkey cells (COS cells), rat myeloma cells, and mouse myeloma cells. These cells may also be cells into which an exogenous gene encoding the protein to be expressed has been introduced. Another example of cells is stem cells such as induced pluripotent stem cells (iPS cells) or mesenchymal stem cells (MSCs). Preferably, the cells are Chinese hamster ovary cells or human embryonic kidney cells 293, and more preferably Chinese hamster ovary cells.

[0028] Expression vectors can be used to introduce foreign genes encoding the protein to be expressed into cells. By introducing an expression vector containing DNA encoding the protein to be expressed, expression regulatory sequences (e.g., enhancers, promoters, and terminators), and optionally a selected marker gene into cells, cells containing the foreign gene encoding the protein to be expressed can be created. There are no particular limitations on expression vectors; they can be appropriately selected and used depending on the type of cell and application.

[0029] Any promoter capable of functioning in mammalian cells can be used. Examples include the promoter of the cytomegalovirus (CMV) IE (immediate early) gene, the early promoter of SV40, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, and the promoter and enhancer of Moloney murine leukemia virus. Additionally, the enhancer of the human CMV IE gene may be used in conjunction with the promoter.

[0030] Examples of selectable marker genes include drug resistance genes (neomycin resistance gene, DHFR gene, puromycin resistance gene, blastosidine resistance gene, hygromycin resistance gene, cycloheximide resistance gene), or fluorescent genes (genes encoding green fluorescent protein GFP, etc.).

[0031] There are no particular limitations on the method for introducing expression vectors into cells; for example, calcium phosphate methods, electroporation, liposome methods, gene gun methods, and lipofection methods can be used.

[0032] After introducing the protein to be expressed and a selection marker gene into cells, multiple monoclonal cells expressing the target protein can be selected using known methods. This disclosure can be used to select a clone suitable for suspension culture from a group of monocloned clones. It is particularly preferable to use it to select a clone suitable for perfusion culture.

[0033] In this disclosure, the damage resistance of each cell can be evaluated by passing a portion of cells from multiple monocloned cell clones through a microfluidic channel. After evaluating the cell damage, clones with high damage resistance can be selected for long-term culture. When selecting these high-damage-resistance clones, other evaluation methods can be combined to further select the clones to be used for culture. Alternatively, after selecting high-damage-resistance clones, further selection can be achieved by combining them with other evaluation methods. These other evaluation methods are not limited to known selection methods, but may include combining protein production efficiency or gene expression as indicators. Perfusion culture is preferred as the culture method.

[0034] In this invention, by selecting cells based on an evaluation of damage resistance, it is possible to preferably select cells that can withstand long-term culture. When selecting cells to produce a target protein, by selecting cells that can withstand long-term culture and using these cells to produce the target protein, it becomes possible to produce a large amount of protein.

[0035] The cells used to produce the target protein described above are preferably cells used to produce the protein by perfusion culture. In the present invention, cells with high perfusion culture tolerance can be selected based on the damage tolerance evaluated by the method of the present invention. Cells with high perfusion culture tolerance can be selected as cells that have a high cell viability and a high bleeding rate (an indicator of cell proliferation rate) in perfusion culture. From the perspective of reproducibility, it is preferable for the cell viability rate after damage loading to have a small CV value (Coefficient of Variation) and standard deviation. The CV value is preferably 10% or less, more preferably 5% or less, and particularly preferably 3% or less.

[0036] Furthermore, in this invention, the threshold for the resistance index can be determined by comparing the measurement results of the resistance index of each cell with the culture results.

[0037] Furthermore, in the present invention, cell clones that have a high survival rate after being subjected to damage using the cell selection device according to the present invention can be selected as cell clones with strong resistance to damage.

[0038] Furthermore, in this invention, the cell culture conditions (whirlpool diameter, whip height, number of whips, stirring speed, various gas flow rates, various bubble diameters) can be determined from the resistance index.

[0039] <Devices and systems for selecting cells> The apparatus for selecting cells according to the present invention is Microchannels and A pressure gauge for measuring the pressure upstream of a microchannel, A first pump for delivering the cell suspension is installed upstream of the microchannel, A second pump for supplying cleaning fluid is installed upstream of the microchannel, A piping for supplying the cell suspension to the first pump, and a piping for supplying the washing solution to the second pump, Piping for supplying the liquid from the first pump and the liquid from the second pump to the microchannel, Piping for collecting the liquid discharged from the above microchannels. This includes the present invention. An example of the apparatus of the present invention is shown in Figure 1.

[0040] Microchannels are pathways designed to impart physical stress to cells. Details of the microfluidic channels are as described above in this specification. As described above in this specification, it is preferable to use a microchannel by passing cells through it once or multiple times, then remove the used microchannel, install a new microchannel, and perform a new measurement. In this case, multiple microchannels will be used. Therefore, by further combining multiple microchannels for interchangeable use with the cell selection device according to the present invention, a cell selection device system can be provided.

[0041] Electroforming is a method of manufacturing pipes using electroplating. It involves depositing metal to a desired thickness onto a mold, and then removing this electrodeposited layer from the mold to obtain the electroformed product. By using an ultrafine metal wire as the mold and then removing the ultrafine wire after electrodeposition to the desired thickness, very fine pipes can be manufactured.

[0042] An example of a method for creating a divided channel by combining a metal plate and a grooved metal plate in a way that allows for disassembly and cleaning is described below with reference to Figure 2. Two 50mm square, 15mm thick stainless steel plates (one without grooves, one with grooves) are used as base plates. For the base plate with grooves, a groove 0.12mm wide, 0.12mm deep, and 50mm long is cut. The surfaces of the two base plates are aligned and four screws are tightened to create a metal-to-metal sealing mechanism (Figure 2).

[0043] A pressure gauge is an instrument that measures pressure values ​​to quantify the physical stress on cells. The pressure gauge is not particularly limited as long as it can measure the pressure upstream of the microchannel; commercially available pressure gauges can be used. The pressure gauge can be installed close to the upstream of the microchannel so that the pressure loss within the microchannel can be estimated. By installing it close to the upstream of the microchannel, it can be considered that the pressure within the microchannel has been measured.

[0044] The system includes two pumps: a first pump for delivering the cell suspension and a second pump for delivering the washing solution. Both the first and second pumps are located upstream of the microchannel.

[0045] The piping can include piping for supplying the cell suspension to the first pump, piping for supplying the washing solution to the second pump, piping for supplying the liquid from the first pump and the liquid from the second pump to the microchannel, and piping for recovering the liquid discharged from the microchannel.

[0046] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0047] Example 1: Evaluation of damage resistance <1> Procedure for evaluating damage resistance <1-1> Preparation before damage resistance evaluation Vectors containing nucleic acid sequences encoding IgG1 and IgG4 were constructed, and these vectors were introduced into CHO-DG44 cells to produce CHO-DG44 cells expressing IgG1 (IgG1 cells) and CHO-DG44 cells expressing IgG4 (IgG4 cells). The construction of the vectors and their introduction into the cells were carried out in accordance with Example 2 of Japanese Patent Publication No. 2016-517691. As a result, three monoclonal clones were prepared from different colonies of CHO cells that produce monoclonal antibodies. I purchased human fetal kidney cells 293 (HEK293) from ATCC.

[0048] Damage resistance was evaluated for CHO cells that had been passaged four times in Erlenmeyer flasks. Before performing the damage resistance evaluation, the cell density of each clone was diluted to 1.5 × 10⁶. 6 The cell concentration was adjusted to cells / ml. After the diluted cell suspension was heated to 37°C in an incubator, its damage resistance was evaluated. Similar to CHO cells, HEK293 cells were also evaluated for damage resistance after being passaged four times in Erlenmeyer flasks. Before performing the damage resistance evaluation, the cell density of the clones was diluted to 1.5 × 10⁶. 6 The cell concentration was adjusted to match the cell / mL. After the diluted cell suspension was heated to 37°C in an incubator, its damage resistance was evaluated.

[0049] <1-2> Damage Resistance Evaluation Device (Figure 1) Two syringe pumps (HARVARD PHD ultra 4400) were used for fluid delivery: one for delivering the cell suspension and the other for delivering the washing solution. A pressure gauge (KEYENCE AP-14S) for quantifying EDR was placed upstream (inlet) of the microchannel. The microchannel itself was made of a circular tube with an inner diameter of 0.1 mm (dimensional tolerance ±2%) and a length of 50 mm, manufactured by electroforming.

[0050] The damage load on the cells is measured by aspirating the cell suspension with a syringe, then switching the valve to deliver the cell suspension into the microchannel (delivery rate: 7.7 ml / min). The EDR is estimated from the pressure value during delivery, and is 2.20~2.30 × 10⁻⁶. 9 [W / m 3 Ensure that the value is within the specified range. If the EDR value falls outside this range, replace the microfluidic channel with a new one.

[0051] <1-3>Quantification of Damage Resistance Evaluation Value For sample collection, approximately twice the volume of piping and sensor components is discarded first, and then the actual sampling is performed.

[0052] (Method for measuring cell viability) Cell viability is the ratio of the density of living cells before and after inflicting damage, and is given by the following formula. "Before damage loading" refers to the state before cells are delivered into the microchannels, while "after damage loading" refers to the state after cells have been delivered into the microchannels.

[0053]

number

[0054] (Method for measuring energy dissipation rate) The energy dissipation rate is calculated by substituting the pipe inner diameter (d), the viscosity of the cell suspension (μ), the pipe length (l), and the measured pressure loss ΔP into the following formula. The pressure loss ΔP is the pressure difference between the primary side (inlet side) of the microchannel and the secondary side (outlet side) of the microchannel. The secondary side (outlet side) of the microchannel is at atmospheric pressure, so it was set to 0. Therefore, the pressure loss ΔP was taken to be the same as the measured primary side pressure.

[0055]

number

[0056] ΔP: Pressure loss [Pa] μ: Viscosity [Pa s] l: Pipe length [m] d: Piping inner diameter [m]

[0057] (Method for measuring shear stress) Substitute the viscosity (μ) of the cell suspension and the energy dissipation rate (ε) from Equation 2 into the following formula to determine the shear stress.

[0058]

number

[0059] μ: Viscosity [Pa s] ε: Energy dissipation rate [W / m 3 ]

[0060] (Method for measuring LDH release rate) The LDH release rate was calculated using the following formula.

[0061]

number

[0062] LDH concentration was measured using a Roche Cedex Bio. For shear-loaded samples, the cell suspension was delivered through a microchannel, then centrifuged at 300G for 5 minutes, and the supernatant was measured. For samples without shear load, the samples not delivered through the microchannel were centrifuged at 300G for 5 minutes, and the supernatant was measured. For samples with complete release, TWEEN® 20 was added to the samples not delivered through the microchannel, and vortexing was continued for 30 minutes. After that, the samples were centrifuged at 300G for 5 minutes, and the supernatant was measured.

[0063] Figure 3 shows the results of measuring the variation in pipe inner diameter when the inner diameter of SUS piping and electroformed piping is calculated from the pressure loss during water flow. The measurement was performed by drawing pure water with a syringe and then switching the valve to deliver it into a microchannel at a rate of 3.0 ml / min. The pipe inner diameter (d) was calculated by substituting the measured pressure loss (ΔP), viscosity (μ), pipe length (l), and flow rate (U) into the following formula.

[0064]

number

[0065] ΔP: Pressure loss [Pa] μ: Viscosity [Pa s] l: Pipe length [m] U:Flow rate [m 3 / s ] d: Piping inner diameter [m]

[0066] Furthermore, Figure 4 and Table 1 show the results of measuring the repeatability of SUS piping and electroformed piping using CHO cells. The measurement involved aspirating a cell suspension with a syringe, then switching valves to deliver the cell suspension into a microchannel. At the SUS piping level, cell viability was evaluated by gradually increasing the flow rate to 6.0, 9.0, and 10.0 [ml / min] at all N3 levels. At the electroformed piping level, cell viability was evaluated by gradually increasing the flow rate of N1 to 4.5, 5.0, 5.5, 6.0, and 7.0 [ml / min], and by gradually increasing the flow rates of N2 and N3 to 5.0, 6.0, 6.5, and 6.8 [ml / min]. The N3 experiments were conducted using new microchannels. Regarding cell viability, the CV values ​​for SUS piping ranged from 7.7% to 19.0%, while the CV values ​​for electroformed piping ranged from 1.5% to 2.1%. Electroformed piping showed lower variability in cell viability, and when measurements were taken with different types of piping, the reproducibility was found to be superior.

[0067] [Table 1]

[0068] Damage resistance evaluation values ​​were quantified using the viable cell density measured by Beckman Coulter's ViCell XR (particle detection conditions: 6-50 [μm], 50 images), as the ratio of viable cell density after damage application to viable cell density before damage application (cell viability). To suppress the influence of variability after the process of filling cells into syringes, the process from "aspirating the cell suspension with a syringe" onwards was performed N=3 (3 experiments). To suppress the influence of measurement variability of ViCellXR, ViCell measurements were performed 3 times for each experiment, and cell viability was calculated. The average of these 3 measured cell viability values ​​was used to calculate the damage resistance evaluation value for each experiment. Furthermore, the average of the average damage resistance evaluation values ​​(average values) from the 3 experiments was used for the cell viability in Figure 5.

[0069] <2> Experimental results The damage resistance evaluation value was quantified twice using the above procedure for three types of CHO cell clones. The results are shown in Figure 5. The damage resistance evaluation value was also quantified twice using the above procedure for human embryonic kidney cells 293. The results are shown in Figure 9. The conditions for measuring HEK293 cells were to gradually increase the flow rate to 3.5, 5.5, and 7.7 [ml / min] at the level of electroformed piping (the EDR at this time was 4.1 × 10⁻⁶). 8 , 1.0 × 10 9 , 2.1 × 10 9 [W / m 3 (This is the result), and cell viability was evaluated.

[0070] The ranking of CHO cell resistance was cloneA (64.9%, 57.4%) > cloneB (50.4%, 49.5%) > cloneC (42.7%, 40.2%), demonstrating a reproducible ranking of damage resistance among clones A, B, and C. It was found that cell damage resistance can also be evaluated in human fetal kidney cells 293.

[0071] Example 2: Perfusion Culture <Perfusion culture method> Cell culture was performed using the perfusion culture method with the cell culture apparatus configured as shown in Figure 6. The cells used were the monoclonal cells described above. The culture vessel was a 2L glass container, the culture medium was OptiCHO (product number 12681011) from Gibco, and the filter for separating cells and antibodies was a hollow fiber membrane (F2 RF02PES) from Repligen. The cell seeding density was 3 × 10⁻⁶ 5 The culture medium was set to cells / mL, and the volume was 1L. Fresh medium was supplied and cell suspension was removed from the culture vessel starting from the third day after the start of culture. During the culture period, the cell density was 120 × 10⁶. 6 Cell bleeding was performed as needed to maintain a cell / ml concentration of approximately 180 rpm. The rotation speed of the stirring device was set to 180 rpm from the start of culture until day 10, and then to 220 rpm thereafter. During the culture period, air was supplied from the top of the culture vessel at a flow rate of 0.1 L / min. Pure oxygen was supplied from a 20 μm spagger placed at the bottom of the culture vessel to maintain an oxygen concentration of 33% in the cell suspension within the culture vessel.

[0072] <Evaluation Methods for Perfusion Culture> Samples were collected from the sampling port, and the viable cell density and cell viability were measured using a ViCell XR from Beckman Coulter. In Figure 7, Viability is the ratio of the viable cell density to the total cell density (sum of viable and dead cell densities) recognized on ViCell. Bleeding in this study refers to the procedure of removing excess cells once a day to adjust to the target cell density. In Figure 8, the Bleeding rate is the ratio of the volume of cell suspension removed by the Bleeding procedure to the total volume of culture medium in the culture tank. The higher the proliferation rate of the cells, the higher the Bleeding rate.

[0073] <Results of perfusion culture> Figure 7 shows the results of measuring cell viability in perfusion culture. When clones A, B, and C were perfused in 1L scale, the average cell viability from Day 10 to Day 52 was clone A (97.6%) > clone B (95.4%) > clone C (91.7%).

[0074] Furthermore, Figure 8 shows the measurement results of the Bleeding rate, which is an indicator of cell proliferation rate in perfusion culture. The average Bleeding rate from Day 10 to Day 52 was cloneA (23.0%) > cloneB (14.1%) > cloneC (8.6%), which followed the same trend as the ranking in the damage resistance evaluation.

Claims

1. Introducing cells into a microchannel and passing them through the microchannel, To evaluate the cell's resistance to damage caused by passing through microchannels, and Based on the evaluation of the aforementioned damage resistance, select suspension culture cells. A method for selecting cells, including, By preparing multiple microchannels and exchanging them, we can evaluate the cells' resistance to damage. The tolerance of the inner diameter of the aforementioned plurality of microchannels is less than ±10%, A method in which the microchannel is an electroformed pipe.

2. The method according to claim 1, wherein the damage to the cells is the stress received from the fluid.

3. The method according to claim 1 or 2, wherein the cell's resistance to damage is evaluated based on the state of cell damage.

4. The method according to any one of claims 1 to 3, wherein damage resistance is evaluated based on the cell viability rate relative to the energy dissipation rate.

5. The method according to any one of claims 1 to 3, wherein damage resistance is evaluated based on the rate of lactate dehydrogenase release from cells relative to the energy dissipation rate.

6. The method according to any one of claims 1 to 3, wherein damage resistance is evaluated based on the cell viability against shear stress.

7. The method according to any one of claims 1 to 3, wherein damage resistance is evaluated based on the rate of lactate dehydrogenase release from cells in response to shear stress.

8. The method according to any one of claims 1 to 7, wherein the inner diameter of the microchannel is 10 to 3000 μm.

9. The method according to any one of claims 1 to 8, wherein the cells are Chinese hamster ovary-derived cells or human fetal kidney cells 293.

10. The method according to any one of claims 1 to 9, wherein the selected cells are cells for producing proteins.

11. The method according to any one of claims 1 to 10, wherein the cells are cells obtained by increasing the number of monocloned cells.

12. The method according to claim 10 or 11, wherein the cells for producing protein are cells for producing protein by perfusion culture.

13. (1) Microchannels, A pressure gauge for measuring the pressure upstream of a microchannel, A first pump for delivering the cell suspension is installed upstream of the microchannel, A second pump for supplying cleaning fluid is installed upstream of the microchannel, A piping for supplying the cell suspension to the first pump, and a piping for supplying the washing solution to the second pump, Piping for supplying the liquid from the first pump and the liquid from the second pump to a microchannel, Piping for recovering the liquid discharged from the aforementioned microchannel. A device for selecting suspension culture cells, wherein the microchannel is an electroformed pipe, and (2) Multiple microchannels for replacement and reuse, A device system for selecting suspension culture cells, wherein the internal diameter tolerance of the plurality of microchannels is less than ±10%, and the microchannels are electroformed pipes.

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