Cell detachment method, and device equipped with cell detachment mechanism

Abstract

The present invention addresses the problem of providing a method for detaching adhered cells from a culture container efficiently and with little damage.　Provided is a method for detaching adherent cultured cells from a culture container, the method including: a cell detachment solution treatment step for adding a cell detachment solution to the cell culture container and treating the cells with the cell detachment solution; and a detachment step for detaching the cells from the cell culture container surface by discharging a discharge solution at the cells after the cell detachment solution treatment, wherein the linear velocity at which the discharge solution is discharged in the detachment step is 300 mm / sec to 1500 mm / sec, and the detachment step either discharges the discharge solution in a spot manner at the culture container surface, the center-to-center distance between neighboring spots being 4 mm to 33 mm, or continuously discharges the discharge solution along linear paths at intervals of 4 mm to 33 mm set on the cell culture container surface.

Description

Cell detachment method and device equipped with cell detachment mechanism

[0001] The present invention relates to a method for detaching cells cultured in adhesion from a culture vessel, a method for producing differentiated cells from adherent stem cells, and an apparatus equipped with a cell detachment mechanism.

[0002] In cell-based assays to evaluate the efficacy and safety of drugs, cells are cultured in the presence of a candidate drug and their response to the drug is measured. To accurately measure drug effects in cell-based assays, it is necessary to maintain consistent culture conditions between different test and control groups. However, this is often difficult when comparing the effects of tens of thousands of candidate drugs, such as those in a compound library.

[0003] In recent years, the establishment of technology for producing induced pluripotent stem cells (iPS cells) has made it possible to generate iPS cells from a patient's own cells and then generate disease-related cells from the iPS cells. It is expected that new, highly effective therapeutic drugs will be developed by conducting drug discovery screening using patient-derived cells, which can more faithfully reproduce the pathology of the disease.

[0004] However, when disease-related cells are generated from patient-derived iPS cells and used to measure drug effects, numerous steps, including cell reprogramming, proliferation, and differentiation induction, must be performed over a long period of time from collection of patient cells to drug screening. To generate a large number of homogeneous cells from iPS cells for drug discovery screening requires highly skilled techniques to perform each step efficiently and reproducibly.

[0005] In particular, iPS cells are adherent cells, and stem cells, progenitor cells, and / or differentiated cells obtained through differentiation induction are often also adherent cells. With adherent cells, each step of transferring them to a culture vessel for proliferation, differentiation induction, etc. involves the work of detaching and recovering the cells from the culture vessel.

[0006] For example, when working with established cell lines such as HeLa cells, the cell properties are stable and even single cells can survive for long periods of time. Therefore, cell detachment can be achieved by immersing the cells in a detachment solution containing trypsin or other agents with strong detachment activity. In the detachment solution, cell-to-cell adhesion and cell-to-substrate adhesion are dissociated, resulting in the cells gradually floating in the detachment solution. Furthermore, a cell suspension can be prepared by rocking the culture vessel or adding a protease inactivation solution.

[0007] On the other hand, when detaching stem cells such as iPS cells, their properties are unstable compared to established cell lines such as HeLa cells, making them more susceptible to the effects of the detachment procedure. Therefore, detachment solutions containing Accutase or TrypLE Select, which have relatively weak detachment activity, are often used, rather than trypsin, which has strong detachment activity. Although such detachment solutions weaken the adhesive strength of the cells, most remain attached and do not float. Therefore, after immersion in the detachment solution, a step of physically detaching the cells from the culture vessel using a cell scraper or the like is required to recover the cells that are still adhered (residual adherent cells).

[0008] Here, the procedure of detaching cells from a culture vessel using a cell scraper or the like can have a significant effect on the viability of the cells obtained, etc. Even for experienced experimenters, it is difficult to efficiently and reproducibly recover cells, and the burden on the operator is significant.

[0009] To reduce the burden on experimenters during cell detachment, devices have been developed that automate the process of detaching cells from culture vessels. Patent Document 1 discloses a device that detaches cells by rocking the culture vessel. However, rocking to detach cells requires continuous high-speed rocking over long periods of time and the use of a detachment solution with strong detachment activity, which can cause significant damage to the cells. For example, this can lead to genome mutations, cell membrane degradation, and elution of intracellular proteases, as well as the inevitable loss of stem cell differentiation potential.

[0010] Therefore, there is a need for a new cell detachment method that allows adherent cells to be detached and recovered from a culture vessel efficiently with minimal disturbance, as well as a new method for preparing homogenous cells from the recovered cells to be subjected to cell assays.

[0011] Patent No. 6718303

[0012] An object of the present invention is to provide a method for efficiently detaching adherent cells from a culture vessel with minimal disturbance.

[0013] In order to solve the above-mentioned problems, the present inventors have developed a new device for detaching cells cultured in adhesion. This device is equipped with a discharge unit that discharges a discharge liquid onto the surface of a culture vessel, and can detach cells by discharging the discharge liquid onto adherent cells. After extensive research, the present inventors have found that cells can be efficiently detached by discharging the discharge liquid onto adherent cells at a specific linear velocity. Furthermore, by discharging the discharge liquid onto the surface of the culture vessel in a spot-like and / or linear discharge pattern, they have succeeded in significantly increasing the cell recovery rate and cell survival rate. The present invention is based on the above-mentioned findings and provides the following.

[0014] (1) A method for detaching cells cultured in adhesion culture from a culture vessel, the method comprising: a cell detachment solution treatment step of adding a cell detachment solution to the culture vessel and treating the cells with the cell detachment solution; and a detachment step of discharging a discharge solution onto the cells after the cell detachment solution treatment to detach the cells from the surface of the culture vessel, wherein the linear velocity of the discharge solution in the detachment step is 300 mm / sec to 1500 mm / sec, and the detachment step involves discharging the discharge solution in spots onto the surface of the culture vessel with a center-to-center distance of 4 mm to 33 mm between adjacent spots, or discharging the discharge solution continuously along a linear path set on the surface of the culture vessel at intervals of 4 mm to 33 mm. (2) The method according to (1), wherein the cell detachment solution contains a protease and / or a chelating agent. (3) The method according to (1) or (2), wherein the treatment with the cell detachment solution in the cell detachment solution treatment step is performed for 1 minute to 30 minutes and / or at 15°C to 45°C. (4) The method according to any one of (1) to (3), wherein the culture vessel is rocked in the cell detachment solution treatment step. (5) The method according to (4), wherein the rocking is performed at an amplitude of 2 mm and / or a frequency of 1 Hz to 8.5 Hz. (6) The method according to any one of (1) to (5), wherein the cell detachment solution is removed between the cell detachment solution treatment step and the detachment step. (7) The method according to any one of (1) to (6), wherein in the detachment step, the discharge angle of the discharge solution relative to the surface of the culture vessel to which the cells are adhered is 75 degrees to 85 degrees. (8) The method according to any one of (1) to (7), wherein in the detachment step, the diameter of the outlet for discharging the discharge solution is 0.1 mm to 2.0 mm. (9) The method according to any one of (1) to (8), further comprising a recovery step of recovering the cells after the detachment step. (10) The method according to (9), wherein the recovery step comprises recovering the cells from a plurality of positions, including the surface and submerged portions of the cell suspension containing the detached cells.

[0015] (11) The method according to any one of (1) to (10), wherein the cells are adherent stem cells, progenitor cells, differentiated cells, established cell lines, or immortalized cells. (12) The method according to (11), wherein the stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and neural stem cells. (13) The method according to (12), wherein the stem cells are induced pluripotent stem cells. (14) The method according to (13), wherein the detachment step ejects the ejected solution in spots onto the surface of the culture vessel, with the center-to-center distance between adjacent spots being 4 mm to 20 mm, or ejects the ejected solution continuously along a linear path set on the surface of the culture vessel at intervals of 4 mm to 20 mm. (15) The number of cells per unit area on the surface of the culture vessel is 21.0 x 10 ４ cells / cm ２(16) The method according to any one of (13) to (15), wherein the cell detachment solution contains 2x TrypLE Select. (17) The method according to (11), wherein the differentiated cells are nerve cells or vascular endothelial cells. (18) The method according to (17), wherein the detachment step discharges a solution in spots onto the surface of the culture vessel, with the center-to-center distance between adjacent spots being 23 mm to 33 mm, or wherein the solution is continuously discharged along a linear path set on the surface of the culture vessel at intervals of 23 mm to 33 mm. (19) The method according to (9), or any one of (10) to (18) citing (9), comprising a repeating step of repeating the steps from the cell detachment solution treatment step to the recovery step. (20) A method for producing differentiated cells from adherent stem cells, comprising: a first culture step of culturing the stem cells in an adherent culture vessel in a first culture vessel; a first cell detachment solution treatment step of adding a first cell detachment solution to the first culture vessel and treating the stem cells with the first cell detachment solution; a first detachment step of discharging a first discharge solution onto the stem cells after the first cell detachment solution treatment step to detach the stem cells from the surface of the first culture vessel; a first recovery step of recovering the stem cells after the first detachment step; a differentiation induction step of adding a differentiation inducer to the stem cells recovered in the first recovery step; a second culture step of culturing progenitor cells and / or differentiated cells obtained in the differentiation induction step in a second culture vessel; and adding a second cell detachment solution to the second culture vessel and treating the progenitor cells and / or differentiated cells with the second cell detachment solution. The method includes a second cell detachment solution treatment step, and a second detachment step of discharging a second discharge solution onto the precursor cells and / or the differentiated cells after the second cell detachment solution treatment, thereby detaching the precursor cells and / or the differentiated cells from the surface of the second culture vessel, wherein the linear velocity at which the first discharge solution is discharged in the first detachment step and / or the linear velocity at which the second discharge solution is discharged in the second detachment step is 300 mm / sec to 1500 mm / sec, and wherein in the first detachment step and / or the second detachment step, the discharge solution is discharged in spots onto the surface of the first culture vessel and / or the second culture vessel, with the center-to-center distance of 4 mm to 33 mm between adjacent spots, or the discharge solution is continuously discharged along a linear path set on the surface of the first culture vessel and / or the second culture vessel at intervals of 4 mm to 33 mm.

[0016] (21) The method according to (20), further comprising a second recovery step of recovering the progenitor cells and / or the differentiated cells recovered in the second recovery step, and a seeding step of seeding the progenitor cells and / or the differentiated cells recovered in the second recovery step onto a multiwell plate. (22) The method according to (20) or (21), wherein the stem cells are induced pluripotent stem cells. (23) The method according to any one of (20) to (22), wherein the differentiated cells are nerve cells. (24) An apparatus equipped with a cell detachment mechanism for detaching cells cultured in adhesion, the apparatus comprising: a cell detachment solution adding unit that adds a cell detachment solution to a culture vessel to which the cells have adhered, and a discharge unit that discharges a discharge solution onto the surface of the culture vessel, the discharge unit discharging the discharge solution at a linear velocity of 300 mm / sec to 1500 mm / sec, and a discharge position control means that controls the position at which the discharge solution is discharged onto the surface of the culture vessel. (25) The apparatus according to (24), further comprising a cell recovery unit that recovers the cells detached from the surface of the culture vessel. (26) The device according to (24) or (25), wherein the discharge unit includes a discharge angle control means for adjusting the discharge angle of the discharge solution relative to the surface of the culture vessel. (27) The device according to any of (24) to (26), wherein the cell detachment solution adding unit is capable of rocking the culture vessel. (28) The device according to any of (24) to (27), wherein the discharge unit has a discharge port with a diameter of 0.1 mm to 2.0 mm. (29) The device according to any of (24) to (28), wherein the discharge position control means discharges the detachment solution in spots, and the interval between adjacent spots is 4 mm to 33 mm.

[0017] (30) The device according to any one of (24) to (28), wherein the discharge position control means continuously discharges the detachment solution along linear paths set on the surface of the culture vessel at intervals of 4 mm to 33 mm. (31) The device according to any one of (25) and (26) to (30), wherein the cell recovery unit includes a recovery position control means for controlling a position at which the detached cells are recovered in a cell suspension containing the detached cells. (32) The device according to (31), wherein the recovery position control means recovers the cells from a plurality of positions including the liquid surface and submerged in the cell suspension. (33) The method according to any one of (1) to (23), wherein the device according to any one of (24) to (32) is used. This specification incorporates the disclosure of Japanese Patent Application No. 2022-006552, from which the present application claims priority.

[0018] According to the present invention, adherent cells can be efficiently detached from a culture vessel with minimal damage.

[0019]

[0023] FIG. 1 is a diagram showing the steps of one embodiment of a cell detachment method of the present invention. The cell detachment method of the present invention includes, as essential steps, a cell detachment solution treatment step (S0101) of treating cells with a cell detachment solution and a detachment step (S0104) of detaching the cells, and includes, as optional steps, a cell detachment solution removal step (S0103) and a recovery step (S0106) of recovering the detached cells.

[0024] FIG. 1 is a diagram showing the steps of one embodiment of a differentiated cell production method of the present invention. The differentiated cell production method of the present invention includes, as essential steps, a first culture step (S0201), a first cell detachment solution treatment step (S0202), a first detachment step (S0203), a first recovery step (S0204), a differentiation induction step (S0205), a second culture step (S0206), a second cell detachment solution treatment step (S0207), and a second detachment step (S0208), and includes, as optional steps, a second recovery step (S0210) and a seeding step (S0212). This figure shows the process of treating adherently cultured cells with a cell detachment solution and detaching them with a discharge solution. Figure 3A shows high-density iPS cells with a confluency of 85% or more obtained by adherent culture. Figure 3B shows iPS cells after treatment with 2xTrypLE Select as a cell detachment solution. Figure 3C shows the discharge of a discharge solution onto the surface of a culture vessel. This figure shows the surface of a culture vessel after cells have been detached with a discharge solution. Figure 4A shows the surface of a culture vessel after a discharge solution has been discharged onto multiple linear paths (arrows). Undetached cells remain in areas where the discharge solution has not been discharged (white). Figure 4B shows an enlarged view of an area where cells have been detached by discharging the discharge solution and an area where cells have not been detached. This figure shows a method for discharging a discharge solution. Figure 5A shows a method for continuously discharging a discharge solution onto multiple linear paths at regular intervals (ΔX) while holding a culture vessel at an inclination angle of 10 degrees relative to the horizontal plane. Figure 5B shows linear paths onto which a discharge solution is continuously discharged. The linear paths are arranged at regular intervals (ΔX) from one another. This figure illustrates a method for discharging a liquid in a spot-like manner. Fig. 6A illustrates discharging to eight locations (discharge position A) using an eight-dispensing arm, and discharging to a position (discharge position B) shifted a fixed distance (ΔY) from discharge position A. Fig. 6B illustrates a discharge position obtained by repeating the discharge from discharge positions A and B shown in Fig. 6A at regular intervals (ΔX) in the X-axis direction.This figure shows an example of a dispensing method that combines spot-like dispensing and dispensing onto a linear path. Figure 7-1A shows an example of the positions where spot-like dispensing and dispensing onto a linear path are performed. Figure 7-1B shows an example of the dispensing position on the entire bottom surface of the culture vessel. Figure 7-1C shows the area where iPS cells were detached by dispensing. The arrowheads indicate the area detached by spot-like dispensing, and the arrows indicate the area detached by dispensing onto a linear path. This is a continuation of Figure 7-1. Figure 7-2D shows the surface of the culture vessel before and after detachment of induced neurons by dispensing onto a linear path. The left photograph shows the surface of the culture vessel before detachment, and the right photograph shows the surface of the culture vessel after detachment. Figure 7-2E shows the surface of the culture vessel before and after detachment of induced neurons by combining spot-like dispensing and dispensing onto a linear path. The left photograph shows the surface of the culture vessel before detachment, and the right photograph shows the surface of the culture vessel after detachment. This figure shows a method for recovering detached cells from a culture vessel. Figure 8A shows the position in the liquid where the cell suspension is recovered using recovery method A. Figure 8B shows two positions for collecting cell suspensions using collection method A. Figure 8C shows the liquid surface positions (collection positions 1 and 2) and the submerged position (collection position 3) for collecting cell suspensions using collection method B. Figure 8D illustrates 24 positions for collecting cell suspensions using collection method B. Figures 9A to 9C show the results of detaching induced neurons. Figures 9A to 9C each show the surface of a culture vessel after detaching induced neurons by discharging the cells along a linear path at a predetermined linear velocity. Figure 9A shows the results under condition 5-1. Figure 9B shows the results under condition 5-2. Figure 9C shows the results under condition 5-3. The dotted frame indicates tangled axons. Figure 9D shows the clumping of cells inside the dispensing tip when collecting cells under condition 5-3. Figure 10A shows the relationship between cell viability in a seeded cell suspension and the CV value in a 384-well plate. Figure 10A shows the results of evaluating the relationship between cell viability and CV value. In the figure, "Condition 1" corresponds to condition 6-1 in Example 6, "Condition 2" corresponds to condition 6-2, and "Condition 3" corresponds to condition 6-3. FIG. 10B shows the results of corrected absorbance measurements (multiple 96 wells) under condition 6-3 in Example 6. FIG. 11A shows the results of MAP2 immunostaining of cells obtained under condition 6-2 in Example 6 (cell viability 60%) and cells obtained under condition 6-3 (cell viability 89%).Arrowheads indicate cells in which the cell nucleus (DAPI) can be confirmed, but the MAP2 signal cannot. Figure 11B shows the percentage of MAP2-positive cells in cells obtained under condition 6-2. Figure 11C shows the results of MAP2 immunostaining of cells obtained under condition 6-3. Figure 11D shows the percentage of MAP2-positive cells in cells obtained under condition 6-3. This figure shows the results of analyzing induced neurons cultured after detachment and recovery in Example 8. Figure 12A shows photographed images of neurons in each well. The arrowheads indicate the cell bodies of neurons, and the arrows indicate the axons of neurons. Figure 12B shows the axon length of neurons in each well of a 96-well plate. This figure shows the results of observing vascular endothelial cells induced to differentiate from iPS cells under a fluorescent microscope after immunostaining in Example 9.

[0020] 1. Cell Detachment Method 1-1. Overview A first aspect of the present invention is a method for detaching cells that have been adherently cultured from a culture vessel (hereinafter also referred to as a "cell detachment method"). The cell detachment method of this aspect includes a cell detachment solution treatment step and a detachment step. According to the cell detachment method of this aspect, cells that have been adherently cultured can be efficiently detached from the culture vessel.

[0021] 1-2. Definition of Terms The following terms frequently used in this specification are defined. As used herein, "detachment" refers to the physical separation of cells from the surface of a culture vessel. Cells may be detached in single cell units or cell clump units. Examples of detachment include physical detachment, enzymatic detachment, chemical detachment, and any combination thereof.

[0022] As used herein, "physical detachment" refers to a method of detaching cells by applying a physical stimulus to the entire or surface of a culture vessel containing cells or to the culture solution. Examples of such methods include mechanical detachment in which the surface of the culture vessel is scraped using a cell detachment means such as a cell scraper, a method in which a liquid is ejected onto the surface of the culture vessel (also known as "splashing"), a method in which the culture vessel is rocked or vibrated, and ultrasonic treatment.

[0023] As used herein, "enzymatic detachment" refers to a method of detaching cells using an enzyme with cell detachment activity. Examples of enzymes used for enzymatic detachment include proteases such as trypsin, TrypLE Select, collagenase, accutase, pronase, papain, and dispase. There are no particular restrictions on the protease used for enzymatic detachment; however, when detaching stem cells such as iPS cells, proteases with relatively low activity (e.g., TrypLE Select, collagenase, accutase) are preferred.

[0024] As used herein, "chemical detachment" refers to a method of detaching cells using a compound such as a chelating agent. The compound is preferably capable of binding calcium ions and magnesium ions. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), and 1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Examples other than chelating agents include phosphate salts containing phosphate ions and carbonate salts containing carbonate ions, which bind calcium ions and magnesium ions. For example, detergents such as phosphate buffer solutions can be used.

[0025] Physical, enzymatic, and chemical detachment can also be used in combination with two or more of these detachment methods. For example, when enzymatic and chemical detachment are combined, a protease such as trypsin can be combined with a chelating agent such as EDTA. For example, commercially available cell detachment agents such as trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technologies), and Accumax (manufactured by Stemcell Technologies) can also be used. Furthermore, after weakening the adhesion between the cells and the surface of the culture vessel by enzymatic and / or chemical detachment, physical detachment can be performed using a cell scraper, splashing, or the like.

[0026] As used herein, the term "culture vessel" refers to any vessel capable of culturing cells, regardless of its material, shape, size, etc. Specific materials for the culture vessel include synthetic resin, natural resin, glass, plastic, metal, etc. Specific shapes of the culture vessel include polygonal prisms such as triangular prisms, cubes, and rectangular parallelepipeds, hemispheres, and cylinders (e.g., those with a circular bottom), with those with a flat bottom (e.g., rectangular or circular) being preferred. Examples of culture vessels include petri dishes, culture flasks, plates, trays, and chips. The plate may be a multiwell plate. A "multiwell plate" refers to a plate containing multiple wells (i.e., two or more wells). Examples of suitable wells include, but are not limited to, 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates, 192-well plates, 384-well plates, and 1536-well plates. Examples of chips include organ-on-a-chips and biofunctional chips.

[0027] The surface of a culture vessel can be coated to promote cell adhesion and cell proliferation. A coating agent is used to coat the culture vessel. Examples of coating agents that can be used include laminin, fibronectin, vitronectin, osteopontin, entactin, collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, and collagen VI), gelatin, poly-L-ornithine, poly-D-lysine, poly-L-lysine, Matrigel (registered trademark), Synthemax (registered trademark) II-S matrix, and the like.

[0028] As used herein, "adherent cells" or "adherent cells" refers to cells that grow while attached to a culture vessel, as opposed to suspension cells that grow suspended in a medium. Adherent cells can be adherent eukaryotic cells, such as insect cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). Adherent mammalian cells are preferred. Examples of adherent cells include adherent stem cells, progenitor cells, differentiated cells, established cell lines, or immortalized cells.

[0029] As used herein, "stem cells" refer to cells that have the ability to self-renew and pluripotency (the ability to differentiate into various cells). Examples of adhesive stem cells as used herein include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MS cells), and neural stem cells.

[0030] As used herein, "progenitor cells" refer to cells at an intermediate differentiation stage between stem cells and differentiated cells. Progenitor cells, as used herein, include cells induced from stem cells and immature cells prior to terminal differentiation into differentiated cells. Specific examples of progenitor cells include neural progenitor cells, cardiovascular progenitor cells, cardiac progenitor cells, common myeloid progenitor cells, T progenitor cells, satellite cells, pancreatic progenitor cells, osteoblasts, and the like.

[0031] As used herein, "differentiated cells" refer to cells that do not further differentiate into different cell types under normal physiological conditions. As used herein, differentiated cells may or may not have the ability to self-renew. Examples of differentiated cells include preneuronal cells, neurons, vascular endothelial cells, vascular wall cells, cardiomyocytes, epithelial cells, fibroblasts, astrocytes, glial cells, hepatocytes, pancreatic islet cells, mesenchymal cells, muscle cells, blood cells, and any combination thereof. Differentiated cells may be differentiated cells induced from stem cells such as iPS cells. Neurons induced from iPS cells are particularly referred to herein as "induced neurons" (e.g., "iN" in Japanese Patent No. 6473077). Differentiated cells may be of either a single cell type or multiple cell types. Examples of established cell lines include CHO cells, COS cells, Vero cells, HEK293 cells, HeLa cells, and NIH3T3 cells. Examples of immortalized cells include RPTEC / TERT1 (immortalized renal proximal tubule epithelial cells), TELOHAEC (aortic endothelial cells), and the like.

[0032] "iPS cells" are cells obtained from somatic cells through induction treatment and have totipotency similar to that of ES cells. iPS cells can be obtained from various types of cells using various methods. They are typically produced by introducing four reprogramming factors, OCT3 / 4, SOX2, KLF4, and C-MYC proteins, into somatic cells. iPS cells are adhesive and have the property of growing while forming colonies, making them less likely to detach than normal cells.

[0033] 1-3. Method The cell detachment method of this embodiment includes a cell detachment solution treatment step and a detachment step as essential steps, and a cell detachment solution removal step and / or a recovery step as optional steps. The cell detachment method of this embodiment may also include a repeat step of repeating the steps from the cell detachment solution treatment step to the recovery step. Each step will be described in detail below.

[0034] (Cell detachment solution treatment step) In this embodiment, the cell detachment solution treatment step is a step of adding a cell detachment solution to a culture vessel and treating the cells with the cell detachment solution.

[0035] As used herein, a "cell detachment solution" refers to a liquid containing at least one component having cell detachment activity (hereinafter referred to as a "cell detachment component"). The cell detachment component contained in the cell detachment solution may be a component used for enzymatic and / or chemical detachment. For enzymatic detachment, proteases such as trypsin, TrypLE Select, collagenase, accutase, pronase, papain, and dispase, and / or nucleases such as DNase I, may be used. For chemical detachment, chelating agents (e.g., EDTA or EGTA) or salts capable of binding calcium ions or magnesium ions (e.g., phosphate or carbonate) may be used. Phosphate-buffered saline (PBS) may also be used as an aqueous solution containing phosphate. Multiple cell detachment components may also be combined; for example, CTK may be used as a detachment agent containing trypsin and collagenase.

[0036] The concentration of the cell detachment component in the cell detachment solution is not limited. For example, in the case of TrypLE Select, 2x TrypLE Select / PBS, which is obtained by diluting 10x TrypLE Select 5-fold with PBS, or 0.5x TrypLE Select / NbM, which is obtained by diluting 1x TrypLE Select 2-fold with Neurobasal Plus Medium, can be used. For example, when the cells are induced pluripotent stem cells, a cell detachment solution containing 2x TrypLE Select can be used. Furthermore, as a chelating agent, a buffer containing 0.1 mM or more EDTA (e.g., 1 mM EDTA / PBS or 0.5 mM EDTA / PBS) or a buffer containing 0.1 M EGTA can be used.

[0037] In one embodiment, the cell detachment solution may contain, in addition to the above-mentioned cell detachment components, a ROCK-specific inhibitor such as Y-27632. Addition of a ROCK-specific inhibitor can prevent cell death of stem cells such as iPS cells.

[0038] The types and concentrations of components of the cell detachment solution can be selected appropriately depending on the type of cells to be detached. For example, 2x TrypLE Select / PBS containing 10 nM Y-27632 can be used to detach iPS cells. For example, a detachment solution consisting of 1x TrypLE Select and Neurobasal plus Medium mixed at an 8:7 ratio can be used to detach neurons such as induced neurons.

[0039] The time for treating cells with the cell detachment solution in the cell detachment solution treatment step may be appropriately set depending on the activity and concentration of the cell detachment component, and may be, for example, 30 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, 1 hour or more and / or 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, e.g., 1 minute to 2 hours, 1 minute to 1 hour, 1 minute to 30 minutes, 10 minutes to 20 minutes, or 15 minutes to 18 minutes.

[0040] The temperature at which the cells are treated with the cell detachment solution in the cell detachment solution treatment step may be any temperature at which the cell detachment component has detachment activity and which does not significantly affect the quality of the cells, and may be, for example, 15°C to 45°C, 20°C to 42°C, 25°C to 40°C, 30°C to 39°C, 35°C to 38°C, or 36°C to 37°C.

[0041] In one embodiment, the culture vessel can be rocked in this step. The rocking conditions are not particularly limited as long as they can further promote cell detachment. The amplitude of the rocking is not limited, and may be, for example, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, or 20 mm or more, and / or 100 mm or less, 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, or 30 mm or less, for example, 1 mm to 4 mm, 2 mm, or 3 mm. The frequency of the rocking is not limited, and may be, for example, 0.5 Hz or more, 1 Hz or more, 2 Hz or more, 3 Hz or more, or 4 Hz or more, 40 Hz or less, 30 Hz or less, 20 Hz or less, 10 Hz or less, or 5 Hz or less, for example, 1 Hz to 10 Hz or 1 Hz to 8.5 Hz.

[0042] The confluency of the cells subjected to the cell detachment solution treatment step is not limited, but is preferably within a range that allows a sufficient amount of cells to be recovered and maintains good cell growth. Specifically, for example, it may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more, and / or less than 100%, less than 99%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, or less than 92%. Preferably, the confluency is 80% or more but less than 100%, and may be, for example, 80% to 98%, 85% to 95%, or 90% to 92%. Specific cell density ranges corresponding to the above confluency ranges include 1 x 10 ４ cells / cm ２ ~100 x 10 ４ cells / cm ２ , 5 x 10 ４ cells / cm ２ ~80 x 10 ４ cells / cm ２ , 10 x 10 ４ cells / cm ２ ~60 x 10 ４ cells / cm ２ , or 20 x 10 ４ cells / cm ２ ~40 x 10 ４ cells / cm ２ An example of a preferred cell density range is 21 x 10 ４ cells / cm ２ ~35 x 10 ４ cells / cm ２ In one embodiment, the cells are stem cells (e.g., induced pluripotent stem cells) and have a cell density range of 21 x 10 ４ cells / cm ２ It may be more than that.

[0043] In one embodiment, this step can be performed by a technician.

[0044] In another embodiment, this step can be carried out using an apparatus (for example, an apparatus equipped with a cell detachment mechanism described in the third aspect below).

[0045] Although the adhesive force between the cells and the surface of the culture vessel is reduced by the cell detachment solution treatment step, it is preferable that some or all of the cells are not completely detached from the surface of the culture vessel.

[0046] (Cell detachment solution removal step) In this embodiment, the cell detachment solution removal step is a selective step of removing the cell detachment solution between the cell detachment solution treatment step and the detachment step described below, for the purpose of removing or inactivating cell detachment components. If the method of this embodiment includes this step, it is preferable because it can prevent the cell detachment components from acting in subsequent steps and damage to the cells.

[0047] The method for removing the cell detachment solution is not particularly limited, and any method may be used as long as the cells are not removed together with the solution. For example, the solution may be removed by suction or decantation.

[0048] The method for inactivating the cell detachment component can be appropriately selected from known methods depending on the type of cell detachment component. For example, if the cell detachment component is a protease, a protease inactivating solution such as a protease inhibitor (e.g., trypsin inhibitor) or a serum-containing medium may be added.

[0049] In one embodiment, this step can be performed by a technician.

[0050] In another embodiment, this step can be carried out using an apparatus (for example, an apparatus equipped with a cell detachment mechanism described in the third aspect below).

[0051] (Detachment Step) In this embodiment, the detachment step is a step of ejecting a discharge liquid onto the cells after the cell detachment solution treatment step, thereby detaching the cells from the surface of the culture vessel.

[0052] As used herein, the term "discharge liquid" refers to a liquid discharged onto cells whose adhesive strength has weakened during the cell detachment solution treatment process (sometimes referred to herein as "residual adherent cells"). The type of discharge liquid is not particularly limited. While the same cell detachment liquid as described above can be used, it is preferable that the liquid does not contain degradative enzymes such as proteases. For example, a buffer solution such as PBS or any medium can be used. The medium is not particularly limited, and commercially available media can be used. Examples include DMEM medium, Ham's F12 medium, DMEM / F12 medium, McCoy's 5A medium, Eagle's MEM medium, αMEM medium, MEM medium, RPMI 1640 medium, Iscove's modified Dulbecco's medium, MCDB 131 medium, William's medium E, IPL 41 medium, and Fischer's medium. In one embodiment, a discharge liquid having the same composition as the cell detachment solution described above is used.

[0053] In the peeling step, the linear velocity at which the discharged liquid is discharged is 300 mm / sec to 1500 mm / sec. In this specification, "linear velocity" refers to the velocity at which the discharged liquid passes through the discharge port. The linear velocity of the discharged liquid can be calculated by dividing the amount of the discharged liquid passing through the discharge port per unit time by the cross-sectional area of ​​the discharge port. The linear velocity at which the discharged liquid is discharged in the peeling step may be 300 mm / sec to 1500 mm / sec, for example, 300 mm / sec to 1400 mm / sec, 350 mm / sec to 1300 mm / sec, 400 mm / sec to 1200 mm / sec, or 500 mm / sec to 1100 mm / sec, and is preferably 550 mm / sec to 1050 mm / sec. Further examples of the linear speed include 400 mm / sec to 700 mm / sec, or 500 mm / sec to 600 mm / sec, 700 mm / sec to 1000 mm / sec, or 800 mm / sec to 900 mm / sec, 700 mm / sec to 1100 mm / sec, or 800 mm / sec to 1000 mm / sec, or 800 mm / sec to 1200 mm / sec, or 900 mm / sec to 1100 mm / sec.

[0054] In one embodiment, the cells are stem cells (e.g., induced pluripotent stem cells), and the linear velocity at which the discharge solution is discharged in the detachment step is 300 mm / sec to 1500 mm / sec, preferably 400 mm / sec to 1200 mm / sec, 500 mm / sec to 1100 mm / sec, or 550 mm / sec to 1050 mm / sec. Further examples of the linear velocity include 400 mm / sec to 700 mm / sec, 500 mm / sec to 600 mm / sec, 700 mm / sec to 1000 mm / sec, 800 mm / sec to 900 mm / sec, or 800 mm / sec to 1200 mm / sec, or 900 mm / sec to 1100 mm / sec.

[0055] In one embodiment, the cells are nerve cells (e.g., nerve cells induced to differentiate from induced pluripotent stem cells), and the linear velocity at which the discharged liquid is discharged in the detachment step may be 500 mm / sec to 1100 mm / sec, or 550 mm / sec to 1050 mm / sec, or 300 mm / sec to 850 mm / sec, 400 mm / sec to 800 mm / sec, or 400 mm / sec to 700 mm / sec. Further examples of linear velocities include 400 mm / sec to 700 mm / sec, 500 mm / sec to 600 mm / sec, 700 mm / sec to 1000 mm / sec, or 800 mm / sec to 900 mm / sec, 700 mm / sec to 1100 mm / sec, or 800 mm / sec to 1000 mm / sec, or 800 mm / sec to 1200 mm / sec, or 900 mm / sec to 1100 mm / sec.

[0056] In one embodiment of this aspect, the discharge liquid can be discharged in a spot-like manner onto the surface of the culture vessel. "Discharging the discharge liquid in a spot-like manner" (hereinafter also referred to as "spot-like discharge") refers to a discharge method in which the discharge liquid is discharged at the same position for a fixed period of time without changing the discharge position, or a discharge method in which the discharge liquid is discharged at the same position for a fixed period of time, and then the discharge position is moved and discharged for a fixed period of time, and this is repeated. In spot-like discharge, cells within a fixed distance from the position where the discharge liquid is discharged are detached.

[0057] When spot-like discharge is performed in the peeling step, the center-to-center distance between adjacent spots is 4 mm to 33 mm. The "center-to-center distance between adjacent spots" (hereinafter also simply referred to as "center-to-center distance") refers to the distance between multiple positions where the discharge liquid is discharged. The center-to-center distance can be appropriately set within the range of 4 mm to 33 mm. More specifically, the center-to-center distance may be 4 mm or more, 4.5 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, 20 mm or more, 25 mm or more, or 28 mm or more, and / or 33 mm or less, 28 mm or less, 25 mm or less, 20 mm or less, 16 mm or less, 15 mm or less, 14 mm or less, 13 mm or less, 12 mm or less, 11 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5.5 mm or less. In one embodiment, the center-to-center distance may be 4 mm to 30 mm, 4 mm to 28 mm, 4 mm to 25 mm, 4 mm to 22 mm, 4 mm to 21 mm, 4 mm to 20 mm, 5 mm to 19 mm, 6 mm to 18 mm, 7 mm to 17 mm, 8 mm to 16 mm, 9 mm to 15 mm, 10 mm to 14 mm, or 11 mm to 13 mm. In a further embodiment, the center-to-center distance may be 4 mm to 15 mm, 4 mm to 12 mm, 4 mm to 10 mm, 4 mm to 9 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, or 4.5 mm to 5.5 mm. A suitable center-to-center distance can be appropriately selected depending on the type of cells to be detached, the type and concentration of cell detachment components contained in the detachment device used, the linear discharge speed, and the like. For example, when iPS cells are detached using 2×TrypLE Select+10 nM Y-27632 or the like, the center-to-center distance can be 10 mm to 19 mm at a linear velocity of 400 mm / sec to 600 mm / sec, or 4 mm to 18 mm at a linear velocity of 600 mm / sec to 1200 mm / sec.

[0058] In one embodiment, the amount of liquid ejected per spot-like ejection may be 1000 μL or less, 750 μL or less, or 500 μL or less, and / or 20 μL or more, 50 μL or more, or 100 μL, for example, 500 μL, 250 μL, or 100 μL. The ejection time per spot-like ejection may be 10 seconds or less, 5 seconds or less, or 1 second or less, and / or 0.01 seconds or more, 0.1 seconds or more, or 0.5 seconds or more, for example, 1.0 second, 1.5 seconds, or 2.0 seconds.

[0059] In one embodiment of this aspect, the discharge liquid can be continuously discharged onto a linear path set on the surface of the culture vessel (hereinafter also referred to as "discharge onto a linear path"). As used herein, "linear path" refers to a discharge position that indicates a linear shape. "Linear shape" here refers to a single rail-like shape. In discharging onto a linear path, the discharge liquid is continuously discharged onto linear paths set on the surface of the culture vessel at intervals of 4 mm to 33 mm. "Linear paths spaced 4 mm to 33 mm apart" means that different portions of a single linear path are set at intervals of 4 mm to 33 mm, and / or that multiple linear paths are set at intervals of 4 mm to 33 mm. More specifically, the linear paths may be spaced apart at intervals of 4 mm or more, 4.5 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, 20 mm or more, 25 mm or more, or 28 mm or more, and / or 33 mm or less, 28 mm or less, 25 mm or less, 20 mm or less, 16 mm or less, 15 mm or less, 14 mm or less, 13 mm or less, 12 mm or less, 11 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5.5 mm or less. In one embodiment, the linear paths may be spaced 4 mm to 33 mm apart, such as 4 mm to 30 mm, 4 mm to 28 mm, 4 mm to 25 mm, 4 mm to 22 mm, 4 mm to 21 mm, 4 mm to 20 mm, 5 mm to 19 mm, 6 mm to 18 mm apart, 7 mm to 17 mm apart, 8 mm to 16 mm apart, 9 mm to 15 mm apart, 10 mm to 14 mm apart, or 11 mm to 13 mm apart. In a further embodiment, the linear paths are spaced 6 mm to 33 mm apart, with further exemplary ranges including 6 mm to 20 mm, 10 mm to 33 mm, 9 mm to 30 mm, 6 mm to 16 mm, 8 mm to 14 mm, 10 mm to 12 mm, 10 mm to 20 mm, 12 mm to 18 mm, 14 mm to 16 mm, 23 mm to 33 mm, 25 mm to 31 mm, or 27 mm to 29 mm apart.

[0060] The preferred spacing between the linear paths can be selected appropriately depending on the linear velocity of discharge. For example, a spacing of 10 mm to 19 mm can be used for a linear velocity of 300 mm / sec to 850 mm / sec, and a spacing of 4 mm to 10 mm can be used for a linear velocity of 850 mm / sec to 1500 mm / sec. In particular, when using a discharge liquid having the same composition as the cell detachment liquid, enzymatic and / or chemical detachment based on the cell detachment components can proceed simultaneously with physical detachment based on the discharge of the discharge liquid. While the effect of physical detachment can be improved at a higher linear velocity, the effect of enzymatic and / or chemical detachment can be advantageously improved at a lower linear velocity, as the cell detachment liquid remains at the discharge position for a longer period of time. Therefore, a linear velocity that satisfies both effects is preferred.

[0061] Furthermore, when discharging onto a linear path, discharging is performed while moving the position of the discharge port relative to the culture vessel. The moving speed of the discharge port is not limited, but may be, for example, 0.1 mm / sec or more, 0.5 mm / sec or more, 1 mm / sec or more, 2 mm / sec, 5 mm / sec or more, or 10 mm / sec or more, and / or 50 mm / sec or less, 40 mm / sec or less, or 30 mm / sec or less, for example, 20 mm / sec, 22.7 mm / sec, or 25 mm / sec.

[0062] The amount of liquid ejected onto the linear path at one time may be, for example, 0.1 mL to 20 mL, 0.5 mL to 10 mL, or 1 mL to 4.5 mL. The ejection time of one ejection onto the linear path may be, for example, 0.1 seconds to 10 seconds, 0.5 seconds to 5 seconds, or 0.5 seconds to 3 seconds.

[0063] In a further embodiment, the discharged liquid can be discharged by combining the above-mentioned spot-like discharge and linear path discharge. For example, cells can be efficiently detached by alternately discharging the liquid in spot-like form and linear path discharge on the surface of the culture vessel.

[0064] In one embodiment, the cells are stem cells (e.g., induced pluripotent stem cells) and the center-to-center distance between adjacent spots is 4 mm to 20 mm and / or the spacing between linear paths is 4 mm to 20 mm, preferably the center-to-center distance between adjacent spots is 4 mm to 15 mm and / or the spacing between linear paths is 4 mm to 15 mm.

[0065] In a further embodiment, the cells are stem cells (e.g., induced pluripotent stem cells), and the linear velocity of ejecting the ejected liquid, the center-to-center distance between adjacent spots, and the spacing between the linear paths can be selected from any combination of the ranges described above. Examples of such combinations include, but are not limited to, a linear velocity of 500 mm / sec to 1100 mm / sec, a center-to-center distance of 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, or 4.5 mm to 5.5 mm, and a spacing between the linear paths of 6 mm to 33 mm, 9 mm to 30 mm, or 9 mm to 17 mm.

[0066] In one embodiment, the cells are neural cells (e.g., neural cells induced to differentiate from induced pluripotent stem cells), and the center-to-center distance between adjacent spots is 23 mm to 33 mm and / or the spacing between linear paths is 23 mm to 33 mm, preferably the center-to-center distance between adjacent spots is 25 mm to 30 mm and / or the spacing between linear paths is 25 mm to 30 mm.

[0067] In a further embodiment, the cells are neural cells (e.g., neural cells differentiated from induced pluripotent stem cells), and the linear velocity of ejecting the ejected liquid, the center-to-center distance between adjacent spots, and the spacing between the linear paths can be selected from any combination of the ranges described above. Examples of such combinations include, but are not limited to, a linear velocity of 500 mm / sec to 1100 mm / sec, 500 mm / sec to 1000 mm / sec, or 500 mm / sec to 600 mm / sec, a center-to-center distance of 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, or 4.5 mm to 5.5 mm, and a spacing between the linear paths of 6 mm to 33 mm, 6 mm to 16 mm, or 23 mm to 33 mm.

[0068] The total amount of the liquid to be discharged in this step is not limited and may be, for example, 1 mL to 100 mL, 2 mL to 50 mL, 3 mL to 30 mL, 5 mL to 20 mL, or 7 mL to 15 mL.

[0069] When the discharge solution is discharged in this step, the surface of the culture vessel may be held horizontally or at a certain inclination angle relative to the horizontal. The range of the inclination angle of the surface of the culture vessel relative to the horizontal is not limited, and may be, for example, 0 to 45 degrees, 1 to 30 degrees, or 1 to 20 degrees, and is preferably 10 degrees.

[0070] Furthermore, in this step, the discharge angle is not limited. In this specification, the "discharge angle" refers to the angle at which the solution is discharged relative to the surface of the culture vessel to which the cells are adhered, and is expressed in the range of 0 to 90 degrees. Therefore, for example, 95 to 105 degrees is synonymous with 75 to 85 degrees. A specific discharge angle may be 90 degrees (vertical) or an angle inclined from the vertical direction. More specifically, the discharge angle may be, for example, 45 to 90 degrees, 50 to 90 degrees, 55 to 90 degrees, 60 to 90 degrees, 65 to 90 degrees, 70 to 90 degrees, or 75 to 85 degrees, preferably 79.5 to 80.5 degrees or 80 degrees.

[0071] The diameter of the outlet for discharging the discharge liquid is not limited as long as adjacent spots or linear paths do not overlap each other. For example, the diameter may be 0.05 mm or more, 0.1 mm or more, or 0.3 mm or more, and / or 5.0 mm or less, 4.5 mm or less, 4.0 mm or less, 3.5 mm or less, 3.0 mm or less, 2.5 mm or less, or 2.0 mm or less, and is preferably 0.05 mm to 5 mm, 0.05 mm to 4 mm, 0.05 mm to 3 mm, 0.1 mm to 2.0 mm, or 0.3 mm to 1.5 mm.

[0072] In one embodiment, this step can be performed by a technician.

[0073] In another embodiment, this step can be carried out using an apparatus (for example, an apparatus equipped with a cell detachment mechanism described in the third aspect below).

[0074] (Recovery Step) In this embodiment, the recovery step is a selective step of recovering the cells from the culture vessel after the detachment step.

[0075] In this step, the method for recovering cells from the culture vessel is not limited. For example, the cell suspension containing the cells may be recovered by aspiration or decantation. When aspiration is performed, the cell suspension may be collected in a specific direction by tilting the culture vessel.

[0076] The position from which the cells are collected from the cell suspension is not limited. For example, the cells may be collected from the liquid surface of the cell suspension containing the detached cells, from the liquid, or from a plurality of positions including the liquid surface and the liquid.

[0077] The number of cells to be collected in this step is not limited, but may be, for example, 1 x 10 ５ pcs or more, 1×10 ６ 10 or more, or 10 x 10 ６ or more, preferably 15 x 10 ６ Furthermore, the viability of the cells recovered in this step is not limited, but is preferably, for example, 70% or more, 80% or more, or 90% or more, and for example, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0078] In one embodiment, this step can be performed by a technician.

[0079] In another embodiment, this step can be carried out using an apparatus (for example, an apparatus equipped with a cell detachment mechanism described in the third aspect below).

[0080] (Repeating step) In this embodiment, the repeating step is a selective step of repeating the steps from the cell detachment solution treatment step to the recovery step. The cell types detached and recovered in the cell detachment solution treatment step to the recovery step and the repeating step may be the same or different. For example, stem cells may be detached and recovered in the cell detachment solution treatment step to the recovery step, and progenitor cells or differentiated cells obtained by inducing differentiation of the stem cells may be detached and recovered in the repeating step. The number of times this step may be repeated is not limited.

[0081] In one embodiment, this step can be performed by a technician.

[0082] In another embodiment, this step can be carried out using an apparatus (for example, an apparatus equipped with a cell detachment mechanism described in the third aspect below).

[0083] 1-4. Effects According to the cell detachment method of this embodiment, cells can be detached and recovered from a culture vessel with a high recovery rate and viability. For example, iPS cells, progenitor cells, and / or differentiated cells obtained by reprogramming cells collected from a patient can be detached and recovered with a high recovery rate and viability after adhesion culture.

[0084] It is generally known that adherent cells with high cell density are difficult to detach from a culture vessel. According to the cell detachment method of this embodiment, even highly confluent adherent cells can be efficiently recovered from the culture vessel.

[0085] Furthermore, stem cells such as iPS cells recovered by the cell detachment method of this embodiment suffer less damage to the cells, are highly uniform, and maintain their differentiation potential.

[0086] Furthermore, by performing all or part of the steps in this embodiment using an apparatus (for example, an apparatus equipped with a cell detachment mechanism as described in the third embodiment below), cells can be detached and recovered efficiently and reproducibly without requiring skilled techniques from the experimenter.

[0087] 2. Method for Producing Differentiated Cells 2-1. Overview A second aspect of the present invention is a method for producing differentiated cells from adherent stem cells (hereinafter also referred to as "method for producing differentiated cells"). The method for producing differentiated cells of this aspect comprises detaching cultured adherent stem cells by the cell detachment method of the first aspect, and further inducing differentiation to obtain progenitor cells and / or differentiated cells, which are then cultured and detached by the cell detachment method of the first aspect. According to the production method of this aspect, differentiated cells can be efficiently produced from adherent stem cells.

[0088] The method for producing differentiated cells of this embodiment includes a first culturing step, a first cell detachment solution treatment step, a first detachment step, a first recovery step, a differentiation induction step, a second culturing step, a second cell detachment solution treatment step, and a second detachment step as essential steps, and includes a second recovery step and / or a seeding step as optional steps. Each step will be described in detail below.

[0089] (First culture step) In this embodiment, the first culture step is a first culture step in which adherent stem cells are cultured in an adhesion manner in a first culture vessel.

[0090] The stem cells cultured in this step are not limited as long as they are adherent stem cells. For example, they may be iPS cells, ES cells, MS cells, or neural stem cells. In one embodiment, the iPS cells are derived from a human patient. For example, they may be iPS cells derived from a patient with familial AD, iPS cells derived from a patient with sporadic AD, or iPS cells derived from a healthy elderly person.

[0091] In addition, the temperature and CO ２ The culture conditions, such as the concentration, culture period, and medium exchange frequency, are not limited. For example, the culture conditions may be 37°C, 5% CO ２ The cells may be statically cultured at 4°C for 1 to 100 days, 1 to 40 days, for example, 2 to 30 days, 3 to 20 days, 4 to 10 days, or 5 to 8 days, depending on the growth status of the cells, with half of the medium being replaced every two days.

[0092] The medium used for culture can be appropriately selected from known media. For example, it can be prepared by using any liquid medium for animal cell culture as a basal medium and appropriately adding other components (serum, serum replacement reagent, growth factor, etc.) as needed. Examples of basal media that can be used include, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, NBp medium, and mixtures thereof (e.g., DMEM / F12 medium, Neurobasal™ Medium (Thermo Fisher Scientific), or NBpDY medium). Examples of other components that can be added to the basal medium include ROCK-specific inhibitors such as Y-27632 and doxycycline.

[0093] The medium used for the above culture may be a commercially available serum-free medium. Examples include, but are not limited to, STK1 and STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), and Corning Stemgro Human Mesenchymal Stem Cell Medium (Corning). Examples of other components added to the basal medium include albumin, serum, serum replacement reagents, and growth factors.

[0094] Furthermore, if the stem cells to be cultured are iPS cells or ES cells, commercially available primate ES cell media or primate ES / iPS cell media may be used. These media may contain known additives suitable for culturing pluripotent stem cells such as ES cells or iPS cells, such as N2 supplement, B27(R) supplement, insulin, bFGF, activin A, heparin, ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitors, and / or GSK-3 inhibitors.

[0095] (First cell detachment solution treatment step) In this embodiment, the first cell detachment solution treatment step is a step of adding the first cell detachment solution to the first culture vessel and treating the stem cells with the first cell detachment solution. This step can be performed in accordance with the cell detachment solution removal step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0096] (First Detachment Step) In this embodiment, the first detachment step is a step of discharging the first discharge liquid onto the stem cells after the first cell detachment solution treatment step to detach the stem cells from the surface of the first culture vessel. This step can be performed in accordance with the detachment step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0097] (First recovery step) In this embodiment, the first recovery step is a step of recovering stem cells after the first detachment step. This step can be performed in accordance with the recovery step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0098] (Differentiation Inducing Step) In this embodiment, the differentiation inducing step is a step of adding a differentiation inducer to the stem cells recovered in the first recovery step.

[0099] As used herein, the term "induction of differentiation" refers to promoting differentiation of cells with differentiation potential, and includes differentiation to a state in the middle of differentiation such as progenitor cells, and differentiation from progenitor cells to differentiated cells at a more advanced stage. In this process, stem cells are differentiated into progenitor cells and / or differentiated cells.

[0100] As used herein, the term "differentiation inducer" refers to a drug that has the activity of inducing differentiation of stem cells into progenitor cells and / or differentiated cells. In this step, this refers to a drug that has the activity of inducing differentiation of stem cells into progenitor cells and / or differentiated cells. The differentiation inducer may be either a low molecular weight compound or a polymer such as a protein.

[0101] In one embodiment, the differentiation inducer is a neural induction agent. Specific examples of the neural induction agent include Shh protein, NGF protein, Noggin protein, Neurturin protein, NT-3 protein, NT-4 protein, and HGF protein, as well as GSK-3 inhibitors (e.g., CHIR99021), γ-secretase inhibitors (e.g., DAPT), and 17-β-estradiol.

[0102] A specific example of a differentiation induction method is to incubate stem cells in a medium containing a differentiation inducer at 37°C and 5% CO ２ The cells are subjected to static culture under these conditions, and on the second or third day, half or all of the medium is replaced. Depending on the state of cell proliferation, the cells are cultured for 1 to 40 days, for example, 2 to 30 days, 3 to 20 days, 4 to 10 days, or 5 to 8 days, thereby enabling differentiation into progenitor cells and / or differentiated cells to be induced.

[0103] When the method of this embodiment includes a second differentiation induction step after the seeding step described below, this step can also be referred to as the "first differentiation induction step."

[0104] (Second culture step) In this embodiment, the second culture step is a step of culturing the precursor cells and / or differentiated cells obtained in the differentiation induction step in a second culture vessel. This step can be performed in accordance with the first culture step described above. Therefore, a detailed description thereof will be omitted here.

[0105] (Second cell detachment solution treatment step) In this embodiment, the second cell detachment solution treatment step is a step of adding a second cell detachment solution to a second culture vessel and treating the progenitor cells and / or differentiated cells with the second cell detachment solution. This step can be performed in accordance with the cell detachment solution treatment step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0106] (Second Detachment Step) In this embodiment, the second detachment step is a step of discharging a second discharging solution onto the progenitor cells and / or differentiated cells after treatment with the second cell detachment solution, thereby detaching the progenitor cells and / or differentiated cells from the surface of the second culture vessel. This step can be performed in accordance with the detachment step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0107] (Second recovery step) In this embodiment, the second recovery step is a selective step of recovering progenitor cells and / or differentiated cells after the second detachment step. This step can be performed in accordance with the recovery step described in the first embodiment. Therefore, a detailed description thereof will be omitted here.

[0108] The cells or cell aggregates recovered in this step can be suspended before seeding, if necessary. For example, by repeating pipetting at 450 μL / sec or more two or more times (e.g., 10 or more times) to disperse the cells into single cells, a uniform cell suspension can be prepared, which is preferable.

[0109] (Seeding Step) In this embodiment, the seeding step is a selective step of seeding the precursor cells and / or differentiated cells collected in the second collection step into a culture vessel such as a multi-well plate.

[0110] The seeding method in this step is not limited. As is the case with methods commonly used in the field of cell culture, a portion of the solution containing the cell aggregates may be collected using a pipette or the like and seeded into a culture vessel such as a multi-well plate containing new medium. When seeding the cells into new medium, it is preferable to place the culture vessel containing the new medium at a culture temperature and preheat the medium.

[0111] The seeding amount is not limited. It may be determined appropriately taking into consideration the volume of the new medium, the desired cell concentration at the start of culture, the type of target cells to be cultured, and the like. For example, the number of cells per well of a multi-well plate may be 10,000 to 100,000 cells / well, 20,000 to 90,000 cells / well, 30,000 to 80,000 cells / well, or 40,000 to 60,000 cells / well. More specifically, the seeding amount may be 40,000 to 100,000 cells / well for a 96-well plate and 10,000 to 25,000 cells / well for a 384-well plate. Specifically, the total number of cells can be calculated using the following formula (I), and the total volume of the cell suspension can be calculated using formula (II).

[0112] Total number of cells = A × B × C + D (Formula I) (In this formula, "A" is the number of multiwell plates prepared, and "B" is the number of cells seeded per well. "C" is a value determined based on the number of wells contained in one multiwell plate. For example, a predetermined value such as "100" for a 96-well plate or "400" for a 384-well plate can be used. "D" is the number of surplus cells. For example, a value calculated by B × C / 2 can be used.) Total volume of liquid = A × E × C + F (Formula II) (In this formula, "A" is the number of multiwell plates prepared, and "E" is the volume of seeding liquid per well. "C" is a value determined based on the number of wells contained in one multiwell plate. For example, a predetermined value such as "100" for a 96-well plate or "400" for a 384-well plate can be used. "F" is the volume of surplus liquid. For example, a value calculated by E × C / 2 can be used.)

[0113] After seeding the cell suspension in the multi-well plate, it is advisable to gently shake the container so that the seeded cells are dispersed more or less uniformly within the container.

[0114] The precursor cells and / or differentiated cells seeded in this step can be cultured in the same manner as in the first culture step.

[0115] The cells seeded by this step (for example, cells in each well of a multi-well plate) may exhibit uniformity in terms of quality such as cell viability with a CV value of 20% or less, or 15% or less, preferably 10% or less.

[0116] The progenitor cells seeded in this step can become differentiated cells. For example, they can become differentiated cells by overculturing. However, if necessary, a second differentiation induction step in which a differentiation inducer is added may be performed to promote differentiation induction.

[0117] Furthermore, when seeding precursor cells and / or differentiated cells into a culture vessel such as a multi-well plate, the coating agent may be removed from the culture vessel as needed, and the vessel may be washed with PBS or the like.

[0118] 2-3. Effects According to the method for producing differentiated cells of this embodiment, differentiated cells such as nerve cells can be efficiently produced from adhesive stem cells such as iPS cells, ES cells, or MS cells.

[0119] Furthermore, by performing all or some of the steps in this embodiment using an apparatus (e.g., an apparatus equipped with a cell detachment mechanism as described in the third embodiment below), the burden on the experimenter can be reduced and differentiated cells can be produced efficiently and reproducibly.

[0120] According to the method for producing differentiated cells of this embodiment, it is possible to prepare a multiwell plate containing highly uniform cells with a CV value of 10% or less. Use of this multiwell plate enables cell assays with high measurement accuracy and high reproducibility.

[0121] 3. Device Equipped with a Cell Detachment Mechanism 3-1. Overview A first aspect of the present invention is a device equipped with a cell detachment mechanism (hereinafter also referred to as a "device equipped with a cell detachment mechanism"). The device equipped with a cell detachment mechanism of this aspect is equipped with a cell detachment liquid addition unit and a discharge unit. The device equipped with a cell detachment mechanism of this aspect can efficiently detach cells that have been adherently cultured from a culture vessel. The device equipped with a cell detachment mechanism of this aspect can also be referred to as a cell detachment device that detaches cells that have been adherently cultured.

[0122] 3-2. Configuration The cell detachment mechanism-equipped device of this embodiment includes a cell detachment solution addition unit and a discharge unit as essential components, and includes a cell recovery unit as an optional component. The cell detachment mechanism-equipped device of this embodiment can include a differentiation induction unit, a cell seeding unit, a cell observation unit, and / or a transport unit as further optional components. Each unit will be described in detail below.

[0123] (Cell detachment liquid adding section) In the cell detachment mechanism-equipped device of this embodiment, the cell detachment liquid adding section can add a cell detachment liquid to the culture vessel to which cells are adhered.

[0124] The cell detachment solution adding unit may include a pipette (pipette tip) for adding the cell detachment solution. The pipette tip can be connected to a pump such as a syringe pump to aspirate and / or dispense the cell detachment solution.

[0125] In one embodiment, the cell detachment solution addition unit can maintain the culture vessel at a temperature at which the cell detachment component has detachment activity, for example, 15°C to 45°C, 20°C to 42°C, 25°C to 40°C, 30°C to 39°C, 35°C to 38°C, or 36°C to 37°C.

[0126] In one embodiment, the cell detachment solution adding unit can control the temperature of the cell detachment solution added to the culture vessel.

[0127] In one embodiment, the cell detachment solution addition unit can rock the culture vessel to further promote cell detachment. The amplitude of the rocking may be, but is not limited to, for example, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 10 mm or more, or 20 mm or more, and / or 100 mm or less, 90 mm or less, 80 mm or less, 70 mm or less, 60 mm or less, 50 mm or less, 40 mm or less, or 30 mm or less, for example, 1 mm to 4 mm, 2 mm, or 3 mm. The frequency of the rocking may be, but is not limited to, for example, 0.5 Hz or more, 1 Hz or more, 2 Hz or more, 3 Hz or more, or 4 Hz or more, 40 Hz or less, 30 Hz or less, 20 Hz or less, 10 Hz or less, or 5 Hz or less, for example, 1 Hz to 10 Hz or 1 Hz to 8.5 Hz. In a further embodiment, the cell detachment solution addition unit includes a rocking speed control means for controlling the rocking speed of the culture vessel. The oscillation speed range can be selected, for example, from the above-mentioned frequency range.

[0128] (Discharge Unit) In the cell detachment mechanism-equipped device of this embodiment, the discharge unit can discharge a discharge liquid onto the surface of a culture vessel. The discharge unit can discharge a discharge liquid at a linear velocity of 300 mm / sec to 1500 mm / sec. The linear velocity at which the discharge unit discharges a discharge liquid may be, for example, 300 mm / sec to 1400 mm / sec, 350 mm / sec to 1300 mm / sec, 400 mm / sec to 1200 mm / sec, or 500 mm / sec to 1100 mm / sec, and preferably 550 mm / sec to 1050 mm / sec. Further examples of the range of the linear velocity at which the discharge portion discharges the discharge liquid include 400 mm / sec to 700 mm / sec, or 500 mm / sec to 600 mm / sec, 700 mm / sec to 1000 mm / sec, or 800 mm / sec to 900 mm / sec, 700 mm / sec to 1100 mm / sec, or 800 mm / sec to 1000 mm / sec, or 800 mm / sec to 1200 mm / sec, or 900 mm / sec to 1100 mm / sec.

[0129] The discharge part can have a discharge port that discharges the discharge liquid into the culture vessel. The diameter of the discharge port is not limited, but may be, for example, 0.05 mm to 5 mm, 0.05 mm to 4 mm, 0.05 mm to 3 mm, 0.1 mm to 2.0 mm, or 0.3 mm to 1.5 mm. The cross-sectional area of ​​the discharge port is, for example, 0.002 mm. ２ ~20mm ２ , 0.002 mm ２ ~13mm ２ , 0.002 mm ２ ~7mm ２ , 0.01 mm ２ ~4mm ２ , or 0.1 mm ２ ~1.8mm ２ may be.

[0130] The discharge port may be, for example, the tip of a pipette (pipette tip). The discharge port of the pipette or the like can be connected to a pump such as a syringe pump to suck and / or discharge the discharge liquid.

[0131] In one embodiment, the discharge unit includes a discharge position control means. The discharge position control means controls the position and / or angle at which the discharge solution is discharged on the surface of the culture vessel. In a further embodiment, the discharge position control means can discharge the detachment solution in a spotted manner. The spacing between adjacent spots may be 4 mm or more, 4.5 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, 20 mm or more, 25 mm or more, or 28 mm or more, and / or 33 mm or less, 28 mm or less, 25 mm or less, 20 mm or less, 16 mm or less, 15 mm or less, 14 mm or less, 13 mm or less, 12 mm or less, 11 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5.5 mm or less. In one embodiment, the distance between adjacent spots may be 4 mm to 33 mm, 4 mm to 30 mm, 4 mm to 28 mm, 4 mm to 25 mm, 4 mm to 22 mm, 4 mm to 21 mm, 4 mm to 20 mm, 5 mm to 19 mm, 6 mm to 18 mm, 7 mm to 17 mm, 8 mm to 16 mm, 9 mm to 15 mm, 10 mm to 14 mm, or 11 mm to 13 mm. In a further embodiment, the distance between adjacent spots may be 4 mm to 15 mm, 4 mm to 12 mm, 4 mm to 10 mm, 4 mm to 9 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, or 4.5 mm to 5.5 mm. In a further embodiment, the discharge position control means can continuously discharge the detachment solution along a linear path set on the surface of the culture vessel. The spacing between the linear paths may be, for example, 4 mm or more, 4.5 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, 20 mm or more, 25 mm or more, or 28 mm or more, and / or 33 mm or less, 28 mm or less, 25 mm or less, 20 mm or less, 16 mm or less, 15 mm or less, 14 mm or less, 13 mm or less, 12 mm or less, 11 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5.5 mm or less.In one embodiment, the spacing between the linear tracks may be 4 mm to 33 mm, 4 mm to 30 mm, 4 mm to 28 mm, 4 mm to 25 mm, 4 mm to 22 mm, 4 mm to 21 mm, 4 mm to 20 mm, 5 mm to 19 mm, 6 mm to 18 mm, 7 mm to 17 mm, 8 mm to 16 mm, 9 mm to 15 mm, 10 mm to 14 mm, or 11 mm to 13 mm. In a further embodiment, the spacing between the linear tracks is 6 mm to 33 mm apart, with further exemplary ranges including spacing between 6 mm to 20 mm, 10 mm to 33 mm, 6 mm to 16 mm, 8 mm to 14 mm, 10 mm to 12 mm, 10 mm to 20 mm, 12 mm to 18 mm, 14 mm to 16 mm, 23 mm to 33 mm, 25 mm to 31 mm, or 27 mm to 29 mm. The discharge angle may be between 0 and 45 degrees, between 1 and 30 degrees, or between 1 and 20 degrees relative to the vertical direction.

[0132] The discharge unit may include a means for controlling the temperature of the discharged liquid.

[0133] (Cell Recovery Section) In the cell detachment mechanism-equipped device of this embodiment, the cell recovery section can recover the cells detached from the surface of the culture vessel.

[0134] The cell collection unit may be a pipette (pipette tip) connected to a pump such as a syringe pump. For example, the cell suspension can be aspirated using a unit having the same configuration as the above-mentioned discharge unit.

[0135] In one embodiment, the cell recovery unit may include a recovery position control means. The recovery position control means controls the position at which cells are recovered in the cell suspension containing the detached cells. The recovery position control means may recover cells from a plurality of positions including the liquid surface and submerged in the cell suspension.

[0136] In a further embodiment, the cell recovery unit may include a means for separating the recovered cells from the recovery solution. An example of the means for separating the cells from the recovery solution is a centrifuge. The cell recovery unit can add new medium to the separated cells and, if necessary, perform pipetting until the cells are monodispersed.

[0137] (Differentiation Inducing Section) In the cell detachment mechanism-equipped device of this embodiment, the differentiation inducing section can add a differentiation inducer to cells such as stem cells recovered in the cell recovery section.

[0138] (Cell Seeding Section) In the device equipped with a cell detachment mechanism of this embodiment, the cell seeding section calculates the total cell number and total liquid volume of cells such as progenitor cells and / or differentiated cells recovered in the cell recovery section, for example, using the above formula I and formula II, and can prepare a cell suspension for seeding into a culture vessel such as a multi-well plate.

[0139] In one embodiment, the cell seeding unit can remove the coating agent from the culture vessel before seeding the cell suspension into the culture vessel.

[0140] (Transportation Unit) In the cell detachment mechanism-equipped device of this embodiment, the transport unit can transport culture vessels such as multi-well plates between each unit. For example, the transport unit can be equipped with a transport arm as a transport means. The transport arm can grasp the culture vessel and transport it to a predetermined unit.

[0141] In one embodiment, the transport unit may include a transport speed control means that can control the transport speed to an appropriate speed depending on the type of culture vessel being transported (e.g., a 1-well plate or a multi-well plate such as a 6-, 24-, 48-, 96-, 384-, or 1536-well plate) so that the content liquid in the culture vessel does not spill during transport.

[0142] (Cell Observation Unit) In the device equipped with a cell detachment mechanism of this embodiment, the cell observation unit can observe cells in a culture vessel. The cell observation unit can photograph the cells under a microscope, etc. The cell observation unit can also calculate the confluency, measure the cell number, and / or calculate the cell survival rate based on the image obtained by photographing the cells.

[0143] 3-3. Effects According to the cell detachment mechanism-equipped device of this aspect, the cell detachment method of the first aspect and the differentiated cell production method of the second aspect can be carried out efficiently.

[0144] The cell detachment mechanism-equipped device of this embodiment can produce differentiated cells efficiently and reproducibly without requiring skilled techniques from an experimenter. For example, it can produce multi-well plates containing highly uniform cells with a CV value of 10% or less.

[0145] Example 1 Linear velocity of ejecting ejection liquid (Purpose) Using a device equipped with a cell detachment mechanism, the ejection velocity of the ejection liquid that can efficiently detach cells cultured in adhesion is investigated.

[0146] This device has been newly developed as a device that can automatically perform processes such as removal of coating liquid, seeding of cell suspension, cell culture, cell detachment, ejection of ejection liquid, cell recovery, differentiation induction, addition of reagents, and viable cell measurement.

[0147] (Methods and Results) (1) Coating of Culture Vessels The coating of culture vessels was carried out as follows. (a) Preparation of iMatrix-511-Coated Vessel A mixture of 100 μL of 0.5 μg / μL iMatrix-511 (Laminin-511 E8) (Nippi, 892001 / 892002) and 15 mL of PBS was added to a rectangular culture vessel (Greiner bio-one REF: 670180) (long side 127.5 mm x short side 85 mm). The culture vessel was immediately shaken to spread the mixture over the entire culture vessel, and then the vessel was incubated in 5% CO ２ The plate was placed in an incubator at 37°C for at least 1 hour. The mixture was removed before seeding the cells. (b) Preparation of PMS-Coated Vessel: A coating solution (hereinafter referred to as PMS coating solution) prepared by diluting 0.5 mL of poly-L-lysine, 1.0 mL of Matrigel, and 1.0 mL of Synthemax with 47.5 mL of sterile water was added to a rectangular culture vessel (Greiner bio-one REF: 670180) (long side 127.5 mm x short side 85 mm) at a volume of 15 mL. Furthermore, for multi-well plates, the PMS coating solution was added to each well at 20 μL / well for 96 wells and 10 μL / well for 384 wells. The PMS coating solution was removed before seeding the cells.

[0148] (2) Cell culture (iPS cells) 0.25-0.37 x 10 cells were cultured in StemFit medium containing 10 nM Y-27632. ４ 15 mL of cell suspension containing 1000 human iPS cells was seeded onto the iMatrix-511 coated culture vessel prepared in (1) above. ２ The cells were placed in an incubator and cultured at 37°C for 9 days, with the medium replaced every 1 to 3 days, until the cells reached a confluency of 85% or more (21.0 x 10 ４ cells / cm ２ ~35.0 x 10 ４ cells / cm ２ ) high-density iPS cells were obtained (Figure 3A).

[0149] (Induced neurons) iPS cells (30 x 10 ６ The cells were suspended in 15 mL of NBpDY medium, inoculated into a PMS-coated container, and incubated in 5% CO ２ After 4 days, 8 mL of the medium was aspirated, and 8 mL of NBpY medium was added. Differentiation induction culture was continued for 3 days, and induced neurons were obtained.

[0150] (3) Cell Detachment Solution Treatment After confirming confluency in the cell observation area, the medium was removed. 15 mL of PBS was added, and the entire cell was washed by shaking the plate. The PBS was then removed. 15 mL of 2x TrypLE Select (10x TrypLE Select diluted 5-fold with phosphate buffered saline (PBS) to form the detachment solution) was added to the culture vessel, and the plate was shaken to distribute the solution throughout. The plate was then placed in a vibrating incubator at 37°C for 15 minutes, and then vibrated in the Y-axis direction at 2 mm amplitude and 4 Hz for 3 minutes to weaken the adhesion between cells and between the cell substrate (Figure 3B). The plate was then tilted 10 degrees from the horizontal.

[0151] (4) Detachment of iPS cells from the culture vessel The culture vessel treated with the cell detachment solution was placed under the outlet (Fig. 3C, Fig. 5A). The inclination angle of the bottom of the culture vessel relative to the horizontal plane was kept at 10° so that the angle of the discharge solution relative to the surface of the culture vessel to which the cells had adhered was 80° (Fig. 5A). The discharge solution was 1.77 mm2 in cross section. ２The solution was ejected vertically from an ejection port consisting of a dispensing tip (3 mm diameter) (Figure 3C). 2x TrypLE Select / PBS + 10 nM Y-27632 was used as the ejection solution, and the ejection solution was continuously ejected along a linear ejection path set in the Y-axis direction (the direction of the short side of the culture vessel) shown in Figure 5B. The ejection solution was ejected at three linear velocities (147 mm / sec, 565 mm / sec, or 848 mm / sec). The ejection port was moved in the Y-axis direction at a speed of 22.7 mm / sec, for a movement time of 3 seconds, and for a movement distance of 68 mm. After ejection of the ejection solution, the cells were photographed under a microscope. The obtained images were analyzed using image analysis software Image J to determine the presence or absence of cell detachment. Specifically, a threshold brightness was determined based on the brightness of the area where cells were present and the area where cells were not present. By binarizing the image based on this threshold brightness, areas where cells remained and areas where cells had detached were distinguished. When a region where cells were detached was observed around the region where the discharged liquid was ejected, it was judged as cell detachment (+), and when no detached region was observed, it was judged as cell detachment (-). The results are shown in Table 1 below.

[0152] The cells detached from the surface of the culture vessel were collected, and the number of collected cells and the percentage of viable cells among the collected cells (cell viability) were evaluated. Specifically, 25 μL of trypan blue and 25 μL of cell suspension were mixed in a microtube, and the mixture was poured into a hemocytometer to count the number of cells. Dead cells are stained with trypan blue. The cell viability was calculated using the following formula (Equation III): ((total number of cells - number of dead cells) / total number of cells) × 100 (Equation III)

[0153] The results of evaluating the presence or absence of cell detachment and cell viability are shown in Table 1 below.

[0154]

[0155] Of the three linear velocities (147 mm / sec, 565 mm / sec, and 848 mm / sec), iPS cells were detached from the culture vessel in the areas where the discharged liquid was discharged at 565 mm / sec and 848 mm / sec (Figure 4). On the other hand, when the discharged liquid was discharged at a linear velocity of 147 mm / sec, iPS cells were not detached from the culture vessel. Furthermore, the cell viability of all detached iPS cells was 80% or higher.

[0156] Example 2: Distance between linear paths for ejection (Objective) When ejection liquid is continuously ejected onto linear paths onto cells, the distance between linear paths that can efficiently detach cells is investigated.

[0157] (Method and Results) iPS cells or induced neurons were cultured in a culture vessel and treated with a cell detachment solution using the same method as in Example 1. In the examples herein, iN cells described in Japanese Patent No. 6473077 were used as induced neurons. Specifically, the induced neurons were neurons induced to differentiate by administering doxycycline (Dox) to human iPS cells to express the Ngn2 transgene.

[0158] Following treatment with the cell detachment solution, the cells were detached by discharging the solution onto the treated cells. With the bottom of the culture vessel tilted at a 10° angle relative to the horizontal, the linear discharge speed was set to 1000 mm / s, and the spacing between adjacent linear paths (shown as ΔX in Figure 5) was set to 15 mm or 28 mm. The solution was continuously discharged onto multiple linear paths (Figure 5). First, on one half of the bottom of the culture vessel, which was held at a higher tilt, discharging was performed onto six linear paths when discharging at 15 mm intervals (Figure 5B, ΔX = 15 mm), or onto four linear paths when discharging at 28 mm intervals (Figure 5B, ΔX = 28 mm). Next, the culture vessel was tilted at a 10° angle to the opposite side, and similar discharging was performed on the remaining half. A total of 3000 μL of the solution was used for each condition.

[0159] The solution used for discharging iPS cells was 2x TrypLE Select / PBS + 10 nM Y-27632, and the solution used for discharging induced neurons was TrypLE Select (1x) (Life Technologies: REF: 12563-011) + 10 nM Y-27632. Other discharging conditions were the same as those described in (4) of Example 1.

[0160] The ratio of the number of viable cells to the number of recovered cells after ejection of the ejection liquid was calculated under the conditions described in Example 1.

[0161] The results of evaluating the number of recovered cells and cell viability are shown in Table 2 below.

[0162]

[0163] The results shown in Table 2 indicate that sufficient cells were recovered under all conditions, but the optimal spacing between linear paths varies depending on the cell type. For iPS cells, more cells were recovered when ejection was performed at 15 mm intervals compared to 28 mm intervals (Table 2: Comparison of Conditions 2-1 and 2-2). For induced neurons, more cells were recovered when ejection was performed at 28 mm intervals compared to 15 mm intervals (Table 2: Comparison of Conditions 2-3 and 2-4). Because high-density cultured iPS cells are difficult to detach using cell detachment solution alone, it is believed that recovery efficiency can be improved by using an ejection method with narrower spacing between linear paths. For induced neurons, while they are easily detached using cell detachment solution, they also have a tendency to aggregate, so it is believed that recovery efficiency would be improved by increasing the spacing between linear paths.

[0164] Example 3: Cell detachment by combining spot-like discharge and linear path discharge (Purpose) Cells are detached from a culture vessel by a method that combines spot-like discharge and linear path discharge.

[0165] (Method and Results) Using the same method as in Example 1, iPS cells or induced neurons were cultured in a culture vessel, and the cells were treated with a cell detachment solution and then a dispensing solution was dispensed onto them to detach the cells. In this example, dispensing was performed in a combination of spot-like dispensing and dispensing onto a linear path. Each type of dispensing was performed using two different dispensers: spot-like dispensing was performed using dispenser 1 with eight dispensing arms, and dispensing onto a linear path was performed using dispenser 2 with a single dispensing arm.

[0166] Specifically, with the bottom of the culture vessel tilted at a 10° angle relative to the horizontal plane, spot-like dispensing and linear dispensing were performed on the half of the bottom of the culture vessel that was held at a higher position due to the tilt. First, in spot-like dispensing using dispenser 1, eight outlets spaced at 10 mm intervals along the Y axis were used to dispense the liquid at a linear velocity of 200 mm / s or 1000 mm / s (Figure 6A, Dispensing Position A, 1.0 mL per outlet). Then, eight outlets were similarly dispensed at a position 5 mm along the Y axis (Figure 6A, Dispensing Position B). A total of 16 spot-like dispensing locations were performed at six positions along the X axis at 11 mm intervals (Figure 6B). Next, dispenser 2 was used to continuously dispense the liquid onto six linear paths set between the positions where spot-like dispensing was performed, with a linear dispensing velocity of 565 mm / s and a spacing of 11 mm between adjacent linear paths (Figure 7-1A). Next, the culture vessel was tilted 10 degrees to the opposite side, and dispenser 1 was used to dispense the liquid in spots onto the opposite half of the surface, and dispenser 2 was used to dispense the liquid along a linear path in the same manner as above (Figure 7-1B). The conditions for dispensing iPS cells in spots at a linear velocity of 200 mm / sec and 1000 mm / sec are shown as Conditions 3-2 and 3-4 in Table 3 below. The conditions for dispensing induced neurons in spots at a linear velocity of 1000 mm / sec are shown as Conditions 3-5.

[0167] For comparison, spot-like ejection of iPS cells was performed at each position on the X axis, at eight points only at ejection position A, and no ejection was performed at ejection position B. The results are shown in Table 3 below as condition 3-1 (linear velocity of spot-like ejection: 200 mm / sec) and condition 3-3 (linear velocity of spot-like ejection: 1000 mm / sec). Furthermore, the results of ejection of induced neurons only along a linear path, without spot-like ejection, are shown as condition 3-6.

[0168] Under each condition, a total of 1000 μL of the ejected liquid was ejected by dispenser 1, and a total of 3000 μL of the ejected liquid was ejected by dispenser 2. Other ejection conditions were the same as those described in (4) of Example 1. After ejection of the ejected liquid under each condition, the presence or absence of cell detachment was confirmed under a microscope, and the results are shown in Table 3 below.

[0169]

[0170] As shown in Table 3, iPS cells were not detached from the culture vessel at a linear velocity of 200 mm / s. On the other hand, iPS cells were detached from the culture vessel at a linear velocity of 1000 mm / s. When only ejection onto a linear path and spot-like ejection at ejection position A were performed (Table 3, condition 3-3), a sufficient amount of cells was recovered, but some iPS cells remained at locations away from the linear path and ejection position A. In contrast, when ejection onto a linear path was combined with spot-like ejection at ejection positions A and B (Table 3, conditions 3-4 and 3-5), iPS cells and induced neurons were detached from the entire surface (Figures 7-1C, 7-2D, E). Under condition 3-6, induced neurons were detached from the entire surface by ejection onto a linear path alone.

[0171] Example 4: Method for recovering detached cells (Objective) A method for efficiently recovering cells detached from a culture vessel was investigated.

[0172] (Method and Results) Visual observation of the culture vessel after detaching iPS cells from the culture vessel using the same method as in Condition 3-4 of Example 3 revealed that in a culture vessel held at an inclination angle of 10 degrees, the detached cells tended to accumulate near the liquid surface, 40 mm to 60 mm from the position where the liquid surface contacts the bottom of the culture vessel. Based on these observation results, the number of recovered cells and cell viability were investigated when cells were recovered from different positions in the culture vessel. The method for measuring the number of recovered cells and cell viability followed the method described in Example 2.

[0173] The locations at which cells were collected using the collection method implemented in this example are shown in Figure 8. Hereinafter, the collection method shown in Figures 8A and 8B will be referred to as "cell collection method A," and the collection method shown in Figures 8C and 8D will be referred to as "cell collection method B." In cell collection method A, cells were collected from two locations in the liquid using a single dispensing arm. In cell collection method B, cells were collected from a total of 24 locations using an eight-dispensing arm: eight locations on the liquid surface (locations 1 and 2 in Figure 8D) at each of two locations on the X-axis (locations 1 and 2 in Figure 8C), and eight locations in the liquid (bottom of the container; location 3 in Figure 8C) (location 3 in Figure 8D). The number of collected cells and cell viability obtained using collection methods A and B are shown in Table 4 below.

[0174]

[0175] In cell recovery method A, the number of recovered cells was 10 x 10 ６ On the other hand, in cell recovery method B, the number of recovered cells was 24 × 10 ６ This indicates that the detached cells can be efficiently recovered by recovering them from multiple positions, including the surface and submerged portions of the cell suspension.

[0176] Example 5: Detachment of induced neurons from culture vessel and evaluation of viability (Objective) To evaluate the conditions for detaching and recovering induced neurons with a high viability.

[0177] (Methods and Results) (1) Cell Culture High-density iPS cells were detached under the conditions 1-1 (Example 1), 2-1 (Example 2), and 3-4 (Example 3) of the above examples, and recovered by the cell recovery method B of Example 4. The obtained iPS cells (30 × 10 ６ The cells were suspended in 15 mL of NBpDY medium, inoculated into a PMS-coated container, and incubated in 5% CO ２ After 4 days, 8 mL of the medium was aspirated, and 8 mL of NBpY medium was added. Differentiation induction culture was continued for 3 days, and induced neurons were obtained.

[0178] (2) Cell detachment solution treatment The induced neuron culture medium was removed, 15 mL of PBS was added, and the entire cell was washed while shaking the plate, after which the PBS was removed. TrypLE Select (1x) + 10 nM Y-27632 was used as the cell detachment solution for induced neurons. 7 mL of cell detachment solution was added to the culture vessel, and the plate was shaken to distribute the solution throughout. The plate was then placed in a vibrating incubator at 37°C for 3 minutes, and then vibrated in the Y-axis direction at an amplitude of 2 mm and 4 Hz for 2 minutes to weaken the adhesive forces between cells and between the cell substrate. Next, 7 mL of NBpDY medium was added, and the plate was tilted 10 degrees relative to the horizontal.

[0179] (3) Detachment of induced neurons from the culture vessel The culture vessel for induced neurons after the cell detachment solution treatment was placed under the discharge port, and the inclination angle of the bottom of the culture vessel with respect to the horizontal plane was maintained at 10 degrees so that the discharge angle of the discharge solution with respect to the surface of the culture vessel was 80 degrees. ２The solution was ejected vertically from an ejection port consisting of a dispensing tip (3 mm diameter). TrypLE Select (1x) + 10 nM Y-27632 was used as the ejection solution. The ejection solution was ejected at three linear velocities (147 mm / sec, 565 mm / sec, or 903 mm / sec) and continuously ejected onto a linear ejection path set in the Y-axis direction (the direction of the short side of the culture vessel) as shown in Figure 5B. The ejection port moved in the Y-axis direction at a speed of 22.7 mm / sec, for a movement time of 3 seconds, and for a movement distance of 68 mm. After ejection of the ejection solution, the cells were photographed under a microscope. The obtained images were analyzed using the image analysis software Image J in the same manner as in Example 1 to determine the presence or absence of cell detachment. The evaluation results are shown in Table 5 below. The number of surviving cells recovered by cell recovery method B is shown in Table 5 below.

[0180]

[0181] At three linear velocities (147 mm / s, 565 mm / s, and 903 mm / s), induced neurons were detached from the culture vessel in the areas where the ejection solution was discharged at 565 mm / s and 903 mm / s, and a sufficient number of cells were recovered. However, in the area where the ejection solution was discharged at 147 mm / s, induced neurons remained on the surface of the culture vessel. Figures 9A-C show the appearance of the surface of the culture vessel after ejection of the ejection solution. From the results shown in Table 5 and Figures 9A-C, it was found that cells could not be sufficiently detached at a low linear velocity (147 mm / s), while at a high linear velocity (903 mm / s), cells were detached forcefully, resulting in entanglement of axons (Figure 9C, dotted line box). Furthermore, under condition 5-3, cell axons were entangled during cell recovery, resulting in the formation of cell aggregates. Not all of the cells detached with the dispensing tip could be recovered, and a slight decrease in the number of recovered cells was observed compared to condition 5-2 (Figure 9D). These results demonstrate that differences in linear velocity significantly affect the number of cells recovered and their viability.

[0182] Example 6: Evaluation of viability of cells seeded in multi-well plates (Objective) After detachment and recovery, induced neurons are seeded in multi-well plates, and after 4 days of culture, the viability and uniformity of the cells in each well are evaluated using WST-8.

[0183] (Method and Results) Cells were detached and collected under each of the following conditions 6-1 to 6-3, and then seeded onto a multi-well plate.

[0184] (Condition 6-1) High-density iPS cells were detached by the methods of Condition 2-1 (Example 2) and Condition 3-4 (Example 3) in the above examples, and recovered by the cell recovery method B in Example 4. The obtained iPS cells (30 × 10 ６ (number of cells) were suspended in 15 mL of NBpDY medium and cultured. After 4 days, 8 mL of medium was aspirated, and 8 mL of NBpY medium was added, followed by differentiation-inducing culture. After a further 3 days, the induced neurons were detached under conditions 2-4 (Example 2) and 3-5 (Example 3) in the above examples, and the induced neurons were recovered under the same conditions as cell recovery method B in Example 4, and allowed to stand as a cell suspension for 3 hours. The total cell number, number of dead cells, and number of viable cells in the obtained cell suspension were measured, and the dead cell contamination rate and cell viability were calculated (shown as cell viability under condition 6-1 in Table 6 below). This cell suspension was adjusted to 4 million viable cells / mL, and 15 mL of the cell suspension was seeded in 25 μL portions into each well of a 384-well plate and incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 4 days.

[0185] (Condition 6-2) High-density iPS cells were detached by the methods of Condition 2-1 (Example 2) and Condition 3-4 (Example 3) in the above examples, and recovered by the cell recovery method B in Example 4. The obtained iPS cells (30 × 10 ６ (number of cells) were suspended in 15 mL of NBpDY medium and cultured. After 4 days, 8 mL of medium was aspirated, and 8 mL of NBpY medium was injected, followed by differentiation-inducing culture. After a further 3 days, the induced neurons were detached under conditions 2-4 (Example 2) and 3-5 (Example 3) in the above examples, and the induced neurons were recovered under the same conditions as in cell recovery method B in Example 4. The total cell number, number of dead cells, and number of viable cells in the obtained cell suspension were measured, and the rate of dead cell contamination and cell viability were calculated (shown as cell viability under condition 6-2 in Table 6 below). This cell suspension was adjusted to 4 million viable cells / mL, and 15 mL of the cell suspension was seeded in 25 μL aliquots into each well of a 384-well plate, and the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 4 days.

[0186] (Condition 6-3) High-density iPS cells were detached by the methods of Condition 2-1 (Example 2) and Condition 3-4 (Example 3) in the above examples, and recovered by cell recovery method B in Example 4. The obtained iPS cells (30 × 10 ６ The cells (number of cells) were suspended in 15 mL of NBpDY medium and cultured. After 4 days, 8 mL of medium was aspirated, and 8 mL of NBpY medium was added, followed by differentiation-inducing culture. After a further 3 days, the induced neurons were detached under condition 5-2 of Example 5 above, and recovered under the same conditions as cell recovery method B of Example 4. The total cell number, number of dead cells, and number of live cells in the cell suspension were measured, and the dead cell contamination rate and cell viability were calculated (shown as cell viability under condition 6-3 in Table 6 below). This cell suspension was adjusted to 4 million viable cells / mL, and 15 mL of the cell suspension was seeded in 25 μL per well of a 384-well plate, and the cells were incubated at 37°C and 5% CO ２ The cells were cultured in an incubator for 4 days.

[0187] (Measurement of viable cell number by WST) Cell Counting Kit-8 (tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt], Dojindo Chemical Industries) solution (2.5 μL) was injected into each well of the 384-well plate at a rate of 10 μL / sec. The solution was injected from the tip of a dispensing tip positioned 1 mm below the liquid surface. The multiwell plate was incubated at 37°C, 5% CO ２ After incubation in an incubator for 3 hours or more, the absorbance of each well was measured at 450 nm and 656 nm, and the corrected absorbance was measured.

[0188] The average value R and standard deviation σ for the 384 wells were calculated, and the CV value (coefficient of variation) was calculated using the following (Equation IV): CV value (%) = σ / R × 100 (Equation IV)

[0189] The dead cell contamination rate and cell viability before seeding, and the CV value of the cells after seeding are shown in Table 6. The relationship between cell viability and CV value is shown in Figure 10A.

[0190]

[0191] The results shown in Table 6 and Figure 10A indicate that cell suspensions with high cell viability have low CV values. These results demonstrate that seeding a cell suspension with high cell viability into a multi-well plate can reduce the variability in cell quality between wells. Seeding a cell suspension with a cell viability of approximately 70% or higher made it possible to prepare a cell plate with a CV value of 10% or less. After preparing a cell suspension of 4 million cells / mL from the cells obtained under condition 6-3 above, 100 μL of the cell suspension was seeded into each well of a 96-well plate to prepare two cell plates. The cell plate was incubated at 37°C and 5% CO ２ After culturing in an incubator for 4 days, 10 μL of Cell Counting Kit-8 solution was added to each well of the cell plate, and the cells were incubated at 37°C, 5% CO ２ After incubation in an incubator for at least 3 hours, the absorbance of each well was measured at 450 nm and 656 nm, and the corrected absorbance was measured. The average value R and standard deviation σ for the 96 wells were calculated, and the CV (coefficient of variation) was calculated using the above formula IV. From the results in Figure 10B, the CV values ​​(%) for the two 96-well plates were 5.04 and 6.20, respectively. For both 96-well plates, cell plates with extremely low inter-plate variability, with CV values ​​kept below 10%, were prepared.

[0192] Example 7: Obtaining immunostained images and calculating the MAP2 positivity rate (Purpose) The induced neurons obtained under condition 6-2 or condition 6-3 of Example 6 after detachment and recovery are seeded onto a multi-well plate, and after 4 days of culture, the homogeneity of the induced neurons in each well is evaluated by immunostaining.

[0193] (Method) The cell suspension obtained under Condition 6-2 or Condition 6-3 was seeded on a 96-well plate in the same manner as above, and the cell plate was incubated at 37°C and 5% CO ２The cells were cultured in an incubator for 4 days. After washing the 96-well plate with PBS, 100 μL of 4% PFA was added to fix the cells, and the fixation was continued for at least 24 hours in a 4°C refrigerator. The 4% PFA was then removed, and 100 μL of permeabilize buffer was added, followed by incubation at room temperature for 2 hours. The permeabilize buffer was then removed, and the plate was washed three times with PBS. Blocking buffer was then added, and the plate was then incubated at room temperature for 2 hours. Anti-MAP2 antibody (Abcam: ab183830) was diluted with blocking buffer at a concentration of 1:500 as the primary antibody. After removing the blocking buffer, the primary antibody solution was added, and the plate was incubated overnight (12 hours or more) at 4°C. The primary antibody solution was removed, and the wells were washed with PBS. A secondary antibody solution prepared by diluting a secondary antibody (Goat anti-rabbit IgG H&L Alexa Fluor 488 (Abcam: ab150077) in blocking buffer at a concentration of 1:2000) was added, and the wells were left to stand at room temperature for 1 hour in the dark. The secondary antibody solution was removed, and the wells were washed twice with PBS. A solution prepared by diluting DAPI 1000-fold with PBS was added to the wells, and the wells were left to stand at room temperature for 10 minutes in the dark. The solution in the wells was removed, and the wells were washed twice with PBS. The wells were then observed under a fluorescence microscope (Keyence BZ-X810). The total number of cells (number of DAPI-positive cells) and the number of MAP2-positive cells (number of neurons) were then counted, and the MAP2 positivity rate was calculated.

[0194] (Results) The results of MAP2 immunostaining of cells obtained by the method under condition 6-2 are shown in Figure 11A and 11B. Among the cells obtained by the method under condition 6-2, MAP2-positive cells with axons and weakly MAP2-positive or MAP2-negative cells without axons (indicated by arrows in Figure 11A) were observed (Figure 11A). With this method, the proportion of MAP2-positive cells (neurons) to the total cells was only approximately 35% (quantitative results for 12 wells of a 96-well plate; Figure 11B).

[0195] Next, the results of MAP2 immunostaining of cells obtained by the method under condition 6-3 are shown in Figures 11C and 11D. It was found that the majority of cells obtained by the method under condition 6-3 were MAP2-positive cells (neurons) (Figure 11C). Furthermore, MAP2-positive cells (neurons) accounted for more than 95% of the total cells (quantitation results for 12 wells in a 96-well plate; Figure 11D).

[0196] Example 8: Evaluation of homogeneity of induced neurons (Objective) The induced neurons obtained by the method of condition 6-3 in Example 6 after detachment and recovery are seeded onto a 96-well plate, and after 4 days of culture, whether or not the induced neurons in each well retain a homogeneous phenotype is evaluated using the axon length of the neurons as an indicator.

[0197] (Method) The cell suspension was seeded onto a 96-well plate in the same manner as described above using the method of condition 6-3, and the cell plate was incubated at 37°C and 5% CO ２ The neurons were cultured in an incubator for 4 days. Images of the neurons in the wells of the 96-well plate were then taken using an Incucyte live cell analysis system (Sartorius Japan Co., Ltd.). The neurons in the captured images were divided into cell bodies and axons, and the axon lengths were calculated.

[0198] (Results) Figure 12A shows photographs of neurons in each well obtained using the method under condition 6-3. Furthermore, the neurons in the photographs were divided into cell body and axon regions, and axon lengths were calculated. The axon lengths of the neurons in each well are shown in Figure 12B. The mean value R and standard deviation σ of the axon lengths of the neurons excluding the 96 outer wells were calculated, and the CV value (coefficient of variation) was calculated using the above formula IV. The CV value of the axon lengths of the neurons in the 96-well plate was 3.19%. It was demonstrated that the method under condition 6-3 can automatically induce differentiation of iPS cells into uniform and homogeneous neurons, and it was confirmed that the axon lengths, a characteristic of neurons, were also uniformly adjusted within the wells.

[0199] Example 9: Preparation of vascular endothelial cell plate (Objective) The differentiation induction into vascular endothelial cells was investigated using iPS cells detached under the methods of Conditions 2-1 and 3-4 of the Examples and recovered by Cell Recovery Method B of Example 4.

[0200] (Method) High-density iPS cells were detached using the methods in Condition 2-1 (Example 2) and Condition 3-4 (Example 3) in the above Examples, and then recovered using Cell Recovery Method B in Example 4. The number of recovered cells and cell viability are shown. Furthermore, the iPS cells obtained by the above procedure were suspended in 15 mL of medium containing StemFit AK02 (Ajinomoto) supplemented with G418 at a final concentration of 50 μL / mL and 10 nM Y-27632 (Nacalai). The cell suspension was seeded at 650,000 cells / 100 μL into each well of a Matrigel-coated 96-well plate, and incubated at 37°C and 5% CO ２ After the iPS cells had adhered, the medium in the 96-well plate was completely replaced with EBM-2 (Lonza) Clonetics medium, and the plate was incubated again at 37°C and 5% CO ２The cells were differentiated and cultured in an incubator for 3 days. The culture supernatant was removed from the 96-well plate, washed with PBS, and then 100 μL of 4% PFA was added. The cells were fixed for 24 hours or more in a 4°C refrigerator. The 4% PFA was then removed, and the plates were washed twice with Blocking Buffer (a solution prepared by diluting Blocking Solution (manufactured by Nakarai) 5 times with pure water). 50 μL of a primary antibody solution containing anti-CD31 antibody (Mouse Monoclonal Ab9498) at a concentration of 1:500 was added to the 96-well plate with the fixed cells, and the plate was left to stand for 12 hours. The primary antibody solution was then removed, and a blocking buffer containing Donkey anti-mouse IgG (H&L) AlexaFluor® 488 as the secondary antibody at a concentration of 1:500 was added and allowed to stand at room temperature for 2 hours. The secondary antibody solution was removed, and the wells were washed twice with PBS. A 1000-fold diluted solution of DAPI in blocking buffer was added to the wells, and the wells were left to stand at room temperature for 10 minutes in the dark. The solution in the wells was then removed, and the wells were washed twice with PBS. Then, observation was performed using a fluorescence microscope (Keyence BZ-X810) to observe CD31-positive cells, a positive marker for vascular endothelial cells. Furthermore, the number of vascular endothelial cells in the 96-well plate was measured in accordance with the method described above for measuring viable cell counts using WST in Example 6, "(Measurement of viable cell counts using WST)." Specifically, 10 μL of a solution of Cell Counting Kit-8 (tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt], Dojindo Chemical Industries) was injected into each well of the 96-well plate at a rate of 10 μL / sec. The multiwell plate was maintained at 37°C, 5% CO ２ After incubation in an incubator for 3 hours or more, the absorbance of each well was measured at 450 nm and 656 nm, and the corrected absorbance was measured. The average value R and standard deviation σ of the 96 wells were calculated, and the CV value was calculated using the above formula IV.

[0201] <Results> Table 7 below shows the number of recovered iPS cells and the cell viability.

[0202] Both iPS cell lines (iPS cells derived from healthy elderly subjects and iPS cells derived from Alzheimer's disease patients) were confirmed to have cell viability of 90% or higher. iPS cells recovered by the above method were induced to differentiate into vascular endothelial cells. The results of immunostaining for the vascular endothelial cell marker CD31 and observing them under a fluorescent microscope are shown in Figure 13. It was revealed that CD31-positive cells could be obtained from both iPS cell lines. Furthermore, to confirm that the iPS cell-derived vascular endothelial cells prepared by the above method were prepared in a uniform cell number within a 96-well plate, viable cell counts were measured using WST using the above method. The results are shown in Table 8 below.

[0203] The inter-well CV value for iPS cell-derived vascular endothelial cells from healthy elderly subjects was 9.98% (n=288). The inter-well CV value for iPS cell-derived vascular endothelial cells from Alzheimer's disease patients was 6.92%.

[0204] The above results demonstrate that, compared to condition 6-2, the method under condition 6-3 enables automatic induction of differentiation of iPS cells into homogeneous nerve cells and vascular endothelial cells.

[0205] The results of Examples 5 to 9 demonstrate that the method and apparatus of the present invention make it possible to prepare homogeneous and uniform cell plates suitable for cell assays for screening.

[0206] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for detaching cells cultured in adhesion from a culture vessel, comprising the steps of: a cell detachment solution treatment step of adding a cell detachment solution to the culture vessel and treating the cells with the cell detachment solution; and a detachment step in which a discharge liquid is discharged onto the cells after the cell detachment solution treatment to detach the cells from the surface of the culture vessel; Including, The linear velocity at which the discharge liquid is discharged in the peeling step is 300 mm / sec to 1500 mm / sec, and The peeling step includes: The solution is discharged in spots on the surface of the culture vessel, and the distance between the centers of adjacent spots is 4 mm to 33 mm; or The method comprises continuously discharging the discharge solution onto linear paths set on the surface of the culture vessel at intervals of 4 mm to 33 mm.

2. The method of claim 1 , wherein the cell detachment solution comprises a protease and / or a chelating agent.

3. 2. The method according to claim 1, wherein the treatment with the cell detachment solution in the cell detachment solution treatment step is carried out for 1 minute to 30 minutes and / or at 15°C to 45°C.

4. The method according to claim 1 , wherein the culture vessel is rocked during the cell detachment solution treatment step.

5. 5. The method according to claim 4, wherein the shaking is performed with an amplitude of 2 mm and / or a frequency of 1 Hz to 8.5 Hz.

6. The method according to claim 1 , wherein the cell detachment solution is removed between the cell detachment solution treatment step and the detachment step.

7. The method according to claim 1, wherein in the detaching step, the angle of the discharged liquid relative to the surface of the culture vessel to which the cells are adhered is 75 degrees to 85 degrees.

8. 2. The method according to claim 1, wherein in the peeling step, the diameter of the discharge port for discharging the discharge liquid is 0.1 mm to 2.0 mm.

9. The method of claim 1 , further comprising a recovery step of recovering the cells after the detachment step.

10. The method according to claim 9 , wherein the recovering step comprises recovering the cells from a plurality of positions including the surface and submerged portions of the cell suspension containing the detached cells.

11. The method of claim 1 , wherein the cells are adherent stem cells, progenitor cells, differentiated cells, cell lines, or immortalized cells.

12. 12. The method of claim 11, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, and neural stem cells.

13. The method of claim 12, wherein the stem cells are induced pluripotent stem cells.

14. The peeling step The liquid is discharged in spots on the surface of the culture vessel, and the distance between the centers of adjacent spots is 4 mm to 20 mm; or The method according to claim 13, wherein the liquid is continuously discharged onto linear paths set on the surface of the culture vessel at intervals of 4 mm to 20 mm.

15. The number of cells per unit area on the surface of the culture vessel was 21.0 × 10 ４ cells / cm ２ The method according to claim 13 .

16. The method of claim 13, wherein the cell detachment solution comprises 2x TrypLE Select.

17. The method according to claim 11, wherein the differentiated cells are nerve cells or vascular endothelial cells.

18. The peeling step includes: The solution is discharged in spots on the surface of the culture vessel, and the distance between the centers of adjacent spots is 23 mm to 33 mm; or The method according to claim 17, wherein the liquid is continuously discharged onto linear paths set on the surface of the culture vessel at intervals of 23 mm to 33 mm.

19. The method according to claim 9 or claim 10 which cites claim 9, further comprising a repeating step of repeating the steps from the cell detachment solution treatment step to the recovery step.

20. A method for producing differentiated cells from adherent stem cells, comprising: a first culture step of adhesion-culturing the stem cells in a first culture vessel; a first cell detachment solution treatment step of adding a first cell detachment solution to the first culture vessel and treating the stem cells with the first cell detachment solution; a first detachment step of discharging a first discharging liquid onto the stem cells after the first cell detachment liquid treatment step to detach the stem cells from the surface of the first culture vessel; a first recovery step of recovering the stem cells after the first detachment step; a differentiation induction step of adding a differentiation inducer to the stem cells recovered in the first recovery step; a second culture step of culturing the precursor cells and / or differentiated cells obtained in the differentiation induction step in a second culture vessel; a second cell detachment solution treatment step of adding a second cell detachment solution to the second culture vessel and treating the precursor cells and / or the differentiated cells with the second cell detachment solution; and a second detachment step in which a second discharging liquid is discharged onto the precursor cells and / or the differentiated cells after the second cell detachment liquid treatment, thereby detaching the precursor cells and / or the differentiated cells from the surface of the second culture vessel; wherein the linear velocity at which the first discharge liquid is discharged in the first peeling step and / or the linear velocity at which the second discharge liquid is discharged in the second peeling step is 300 mm / sec to 1500 mm / sec; The first peeling step and / or the second peeling step include: The discharge solution is discharged in spots on the surface of the first culture vessel and / or the second culture vessel, and the distance between the centers of adjacent spots is 4 mm to 33 mm; or The method, wherein the liquid is continuously discharged onto linear paths set on the surface of the first culture vessel and / or the second culture vessel at intervals of 4 mm to 33 mm.

21. a second recovery step of recovering the precursor cells and / or the differentiated cells after the second detachment step; and a seeding step of seeding the precursor cells and / or the differentiated cells recovered in the second recovery step onto a multiwell plate; 21. The method of claim 20, further comprising:

22. 21. The method of claim 20, wherein the stem cells are induced pluripotent stem cells.

23. 21. The method of claim 20, wherein the differentiated cells are neuronal cells.

24. An apparatus equipped with a cell detachment mechanism for detaching cells cultured in adhesion, a cell detachment solution adding unit that adds a cell detachment solution to the culture vessel to which the cells have adhered; and a discharge unit that discharges a discharge liquid onto the surface of the culture vessel; Equipped with The device, wherein the discharge unit discharges the discharge liquid at a linear velocity of 300 mm / sec to 1500 mm / sec, and includes a discharge position control means for controlling the position at which the discharge liquid is discharged on the surface of the culture vessel.

25. The apparatus according to claim 24 , further comprising a cell recovery section that recovers the cells detached from the surface of the culture vessel.

26. The device according to claim 24 , wherein the discharge unit includes a discharge angle control means for adjusting the discharge angle of the discharge liquid relative to the surface of the culture vessel.

27. The device according to claim 24 , wherein the cell detachment solution addition unit is capable of rocking the culture vessel.

28. The device according to claim 24, wherein the discharge portion has a discharge opening with a diameter of 0.1 mm to 2.0 mm.

29. 25. The device according to claim 24, wherein the ejection position control means ejects the stripping liquid in spots, with the interval between adjacent spots being 4 mm to 33 mm.

30. 25. The device according to claim 24, wherein the discharge position control means continuously discharges the detachment solution onto linear paths set on the surface of the culture vessel at intervals of 4 mm to 33 mm.

31. The device according to claim 25 , wherein the cell recovery unit includes a recovery position control means for controlling a position at which the cells are recovered in a cell suspension containing the detached cells.

32. The device according to claim 31 , wherein the recovery position control means recovers the cells from a plurality of positions including the liquid surface and submerged in the cell suspension.

33. 25. The method of claim 1, using an apparatus according to claim 24.