Method of cell inoculation

Abstract

The invention relates to a cell inoculation method and a cell culture method. Specifically, the present invention relates to a method for inoculating cells by means of a dispensing machine, which comprises a step for sucking a cell suspension by means of the dispensing machine and a step for discharging the cell suspension to a fixed point in a culture vessel by means of the dispensing machine.

Description

Technical Field

[0001] The present invention relates to a method for cell seeding, and more particularly to a method for cell seeding using a dispenser. Furthermore, the present invention relates to a method for cell culture. Background Art

[0002] In recent years, it has become possible to use stem cells such as human iPS cells to produce differentiated cells that were previously difficult to obtain. Furthermore, there are cases where differentiated cells derived from iPS cells derived from disease patients are used to mimic disease states in vitro, increasing the success rate of drug development and promoting their use as cell products (Sharma, A. et al., CellStem Cell. 2020; 26(3): 309-329).

[0003] Producing differentiated cells derived from human stem cells requires sophisticated techniques and long-term culture, making it difficult to ensure cell quality and reproducibility between experiments. For example, cell density, a key factor in determining the direction of differentiation, is difficult to precisely control, and the properties of the resulting cells vary depending on the skill of the experimenter. Furthermore, even the same skilled technician cannot replicate identical experimental procedures.

[0004] As mentioned above, cell density is a factor that affects the fate and differentiation direction of the cell. In the examples known in the past, it was reported that skeletal muscle myoblasts such as C2C12 would fuse and form muscle fibers due to the increase in cell density (non-patent literature 1). In addition, in recent years, multiple reports have pointed out that the quality of iPS cells expected to be used for drug development and cell medicine will also change significantly according to cell density. As an example, it was reported that cell density can make the expression of transcription factors as undifferentiated indicators different (non-patent literature 2), and that seeding density can have an impact on epigenetic memory, with the result that the differentiation direction changes (non-patent literature 3). In addition, it was also reported that the unevenness of the cell density in the culture container, such as the deviation of local cell density, can make the cell group obtained, i.e. the cell group after differentiation different. Specifically, it is disclosed that when liver cells are induced by iPS cell differentiation, epithelial cells appear at a location with a thin cell density, and the purity of the target cells decreases (non-patent literature 4).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2021-023173

[0008] Non-patent literature

[0009] Non-patent literature 1: Messina, G. et al., Molecular Biology of the Cell. 2005;16: 1469-1480

[0010] Non-patent literature 2: Myers, FB et al., Integr. Biol. 2013; 5: 1495-1506

[0011] Non-patent literature 3: Kim, M.-H. et al., Stem Cell Research 2021; 56: 102534

[0012] Non-patent literature 4: Graffmann, N. et al., PLoS ONE 2018; 13(7): e0200416 Summary of the Invention

[0013] Problems to be solved by the invention

[0014] In order to improve cell quality, the introduction of robots in drug development and cell product manufacturing processes has been reported (Kanda, GN et al., eLife 2022; 11: e77007). In such cell culture using an automated cell culture device, a method for improving the method by avoiding residual bubbles inside the hole when the cell suspension is inoculated into the hole has been reported (Patent Document 1). However, it is still difficult to produce homogeneous cells with good reproducibility, especially for mass production. In addition, in order to obtain homogeneous cells with good reproducibility, it is desirable to develop an inoculation method with small deviation of local cell density in the culture container.

[0015] Therefore, the object of the present invention is to provide a method and means for uniformly seeding cells in a culture vessel. In addition, the object of the present invention is to provide a method and means for culturing the uniformly seeded cells and producing homogeneous cells with good reproducibility, particularly for mass production.

[0016] Methods used to solve problems

[0017] The present inventors discovered that the method used to discharge cells from a dispenser during cell seeding affects the uniformity of cells within the culture vessel. They explored conditions for uniform cell seeding and found that by using an automated system to discharge a uniform cell suspension into a single point in the culture vessel and optionally allowing the cells to rest without shaking the culture vessel, the cell density within the culture vessel can be controlled without local variations, enabling reproducible mass production of homogeneous cells. Furthermore, they found that cells seeded in this manner can be used to reproducibly mass-produce homogeneous differentiated cells.

[0018] For example, the present invention includes the following embodiments.

[0019] [1] A method for cell seeding using a dispenser, comprising:

[0020] A step of aspirating the cell suspension using a dispenser; and

[0021] A step of discharging the cell suspension to a fixed point in the culture container using the dispenser.

[0022] [2] The method according to [1], wherein the suction step and the discharge step are performed once or twice or more respectively.

[0023] [2-1] The method according to [1], wherein the suction step and the discharge step are performed 2 to 4 times respectively.

[0024] [3] The method according to [1] or [2], further comprising the step of allowing the cells to adhere to the culture container by leaving the container still without shaking it after the discharge step.

[0025] [4] The method according to any one of [1] to [3], further comprising a step of stirring the cell suspension before the aspiration step.

[0026] [5] The method according to any one of [1] to [3], further comprising a step of stirring the cell suspension before the aspiration step, and then performing the aspiration step and the discharge step once or twice or more, respectively.

[0027] [6] The method according to [4] or [5], wherein the aspirating step and the discharging step are completed after the stirring step and before the cells in the cell suspension settle.

[0028] [7] The method according to any one of [1] to [6], wherein the fixed point in the culture container is located at the center of the bottom surface of the culture container.

[0029] [8] The method according to any one of [1] to [7], wherein the culture container is a flat-bottomed culture container.

[0030] [8-1] The method according to any one of [1] to [7], wherein the culture container is a quadrilateral flat-bottomed culture container.

[0031] [9] The method according to any one of [1] to [8], wherein the cells are stem cells, preferably iPS cells.

[0032]

[10] The method according to any one of [1] to [9], wherein the dispensing machine is an automated dispensing machine.

[0033]

[11] A method of culturing cells in a culture container, comprising:

[0034] A step of seeding cells in a culture container by the method of any one of [1] to

[10] ; and

[0035] The step of culturing the above cells in the above culture container.

[0036] [11-1] The method according to

[11] , further comprising the step of transferring the culture container to an incubator after the cell inoculation step.

[0037] [11-2] The method according to

[11] , further comprising the step of inducing differentiation of the above-mentioned cells.

[0038]

[12] A method for dispensing a cell suspension using a dispensing machine, comprising:

[0039] A step of aspirating the cell suspension using a dispenser; and

[0040] A step of discharging the cell suspension to a fixed point in the culture container using the dispenser.

[0041]

[13] A method of making a stock of cells, comprising:

[0042] A step of seeding cells in a culture container by the method of any one of [1] to

[10] ; and

[0043] The step of culturing the above cells in the above culture container.

[0044] [13-1] The method according to

[13] further includes the step of transferring the culture container to an incubator.

[0045] [13-2] The method according to

[13] , further comprising the step of inducing differentiation of the above-mentioned cells.

[0046] [13-3] The method according to

[13] , further comprising the step of recovering the cultured cells from the culture container.

[0047]

[14] A cell seeding device comprising a dispenser, a portion for holding a cell suspension, a portion for holding a culture container, and a control unit.

[0048] The control unit is controlled in the following manner:

[0049] The dispensing machine is caused to aspirate the cell suspension held by the cell suspension holding portion.

[0050] The cell suspension sucked into the dispenser is discharged to a fixed point in the culture container held by the culture container holding portion.

[0051]

[15] A cell culture system comprising the cell seeding device described in

[14] , an incubator, a transfer mechanism, and a control unit.

[0052] The control unit is controlled in the following manner:

[0053] The culture container is transferred from the culture container holding portion of the cell seeding device to the incubator by means of the transfer mechanism.

[0054] The cells in the culture container are cultured in the incubator.

[0055]

[16] A procedure for cell seeding using a dispenser, wherein the dispenser performs the following steps:

[0056] a step of aspirating the cell suspension held in the portion holding the cell suspension;

[0057] A step of discharging the cell suspension to a fixed point in the culture container held by a culture container holding portion.

[0058] [16-1] The program according to

[16] , wherein the above-mentioned dispensing machine is caused to perform the above-mentioned suction step and discharge step once or twice or more (for example, 2 to 4 times).

[0059] [16-2] The procedure according to

[16] , wherein, before the aspiration step, the dispenser is further caused to perform a step of stirring the cell suspension.

[0060] [16-3] The procedure according to [16-2], wherein the aspirating step and the discharging step are completed after the stirring step and before the cells in the cell suspension settle.

[0061] [16-4] The procedure according to

[16] , wherein a fixed point in the culture container is set at a central portion of the bottom surface of the culture container.

[0062] Effects of the Invention

[0063] The cell inoculation method, cell culture method, cell inoculation device, and cell culture system of the present invention enable cells to be uniformly inoculated into a culture vessel, resulting in the reproducible mass production of homogeneous cells. Furthermore, when the cultured cells are subjected to differentiation induction, homogeneous differentiated cells can also be reproducibly mass-produced. Therefore, the present invention is useful in fields such as cell culture, drug development, and the manufacture of clinical cell preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from seeding according to condition (i) in Example 1.

[0065] Figure 2 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from seeding under condition (ii) in Example 1.

[0066] Figure 3 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) obtained by seeding under the condition of 10 shaking times in Example 2.

[0067] Figure 4 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from seeding under the conditions of 20 shakes in Example 2.

[0068] Figure 5 The cell density (number of cells per field of view) (A) and a heat map based thereon (B) are shown when the cells were seeded without shaking in Example 2 and when the cells were seeded at a discharge rate of 890 μL / sec in Example 4.

[0069] Figure 6 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) obtained when the cells were seeded with stirring each time in Example 3.

[0070] Figure 7 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 179 μL / sec in Example 4.

[0071] Figure 8Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 358 μL / sec in Example 4.

[0072] Figure 9 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 1078 μL / sec in Example 4.

[0073] Figure 10 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 1315 μL / sec in Example 4.

[0074] Figure 11 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 1780 μL / sec in Example 4.

[0075] Figure 12 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 2520 μL / sec in Example 4.

[0076] Figure 13 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from inoculation at a discharge rate of 4319 μL / sec in Example 4.

[0077] Figure 14 This is a graph showing the CV value (A) and the maximum value / average value (B) obtained by inoculating at each discharge rate in Example 4.

[0078] Figure 15 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from seeding under the conditions of one-point discharge and no oscillation in Example 5.

[0079] Figure 16 Shown are the cell density (number of cells per field of view) (A) and a heat map based thereon (B) resulting from seeding under the conditions of two-point ejection and no oscillation in Example 5.

[0080] Figure 17 The cell density (number of cells per field of view) obtained by seeding under the conditions of one-point discharge and 10 oscillations in Example 5 (A) and a heat map based thereon (B) are shown.

[0081] Figure 18Shown are the cell density (number of cells per field of view) formed by seeding HEK293T cells in Example 6 (A) and a heat map based thereon (B).

[0082] Figure 19A The cell density (nuclear area per field of view) when seeded according to the one-point discharge condition in Example 7 is shown.

[0083] Figure 19B A heat map showing the cell density (nuclear area per field of view) when seeded under the conditions of one-point exclusion in Example 7 is shown.

[0084] Figure 20A The cell density (nuclear area per field of view) when seeded according to the two-point exclusion conditions in Example 7 is shown.

[0085] Figure 20B A heat map showing the cell density (nuclear area per field of view) when seeded based on the two-point exclusion condition in Example 7.

[0086] Figure 21A The MyHC expression level per cell after differentiation induction under the conditions of one-point exclusion in Example 7 is shown (skeletal muscle area / nuclear area per field of view).

[0087] Figure 21B A heat map showing the MyHC expression level per cell after differentiation induction under the conditions of one-point exclusion in Example 7 (skeletal muscle area / nuclear area per field of view).

[0088] Figure 22A The MyHC expression level per cell after differentiation induction under the two-point exclusion conditions in Example 7 is shown (skeletal muscle area / nuclear area per field of view).

[0089] Figure 22B A heat map showing the MyHC expression level per cell (skeletal muscle area / nuclear area per field of view) after differentiation induction under the two-point exclusion conditions in Example 7 is shown.

[0090] Figure 23A The cell density (number of cells per field of view) of C2C12 myoblasts at the time of seeding under the one-point exclusion condition in Example 8 is shown.

[0091] Figure 23B A heat map showing the cell density (number of cells per field of view) of C2C12 myoblasts at the time of seeding under the conditions of one-point ejection in Example 8.

[0092] Figure 24A The cell density (number of cells per field of view) of C2C12 myoblasts at the time of seeding under the two-point exclusion condition in Example 8 is shown.

[0093] Figure 24B A heat map showing the cell density (number of cells per field of view) of C2C12 myoblasts at the time of seeding under the two-point exclusion condition in Example 8.

[0094] Figure 25A The MyHC expression level per cell (skeletal muscle area / nuclear area per field of view) after differentiation induction of C2C12 myoblasts under the conditions of one-point exclusion in Example 8 is shown.

[0095] Figure 25B A heat map showing the MyHC expression level per cell (skeletal muscle area / nuclear area per field of view) after differentiation induction of C2C12 myoblasts under the conditions of one-point exclusion in Example 8.

[0096] Figure 26A The MyHC expression level per cell (skeletal muscle area / nuclear area per field of view) after differentiation induction of C2C12 myoblasts under the two-point exclusion conditions in Example 8 is shown.

[0097] Figure 26B A heat map showing the MyHC expression level per cell (skeletal muscle area / nuclear area per field of view) after differentiation induction of C2C12 myoblasts under the two-point exclusion conditions in Example 8. DETAILED DESCRIPTION

[0098] The present invention is described in detail below.

[0099] The present invention relates to methods and apparatus for cell seeding, as well as methods and systems for cell culture. Some of these solutions are based on the insight that when seeding cells using a dispenser, the uniformity of cell seeding within the culture vessel can be improved by dispensing the cell suspension into a fixed point within the culture vessel, stirring the cell suspension before aspirating it, not shaking the suspension after dispensing it, and dispensing it at an appropriate rate. Furthermore, when inducing differentiation of the thus seeded cells, homogeneous differentiated cells can be produced in large quantities with good reproducibility.

[0100] As used herein, "cell seeding" refers to the inoculation of cells into a culture medium on or within a culture vessel, a procedure commonly understood in the field of cell culture. Furthermore, "culturing" cells refers to at least one of the following: maintenance of cells in a culture vessel, cell growth, cell differentiation, cell proliferation, and formation of three-dimensional cell aggregates such as spheroids.

[0101] In this specification, regarding cells, "uniform" means that cells in the culture container do not exist in a manner that the number of cells is biased towards some areas, but exist in a uniform number of cells as a whole. For example, it can be judged as follows: after the culture container is divided into two or more areas and the number of cells in each area is obtained, any one or combination of (1) CV value (standard deviation (SD) / mean value), (2) maximum value / mean value (the maximum value (Max) of the number of cells in each area divided by the mean value (Mean)), (3) a heat map made based on the number of cells in each area can be used to judge whether it is uniform. The lower the CV value and the maximum value / mean value, the more uniform it can be judged to be. The smaller the difference in the color depth of the heat map based on visual observation, the more uniform it is, and it can be judged that there is no deviation in local cell density. As an example, for example, the CV value is preferably less than 13%, and the maximum value / mean value (Max / Mean) is preferably less than 130%, but it is ultimately judged whether it is uniform by visually observing the heat map. On the other hand, in this specification, regarding cells, "homogeneous" means that the properties (proliferation ability, differentiation ability, degree of differentiation, characteristics, etc.) of the cells are equal or approximately equal.

[0102] (Cell seeding method)

[0103] In one embodiment, the present invention provides a method for cell seeding using a dispenser, comprising: aspirating a cell suspension using the dispenser; and discharging the cell suspension to a fixed point in a culture container using the dispenser.

[0104] The "cells" that become the object are not particularly limited as long as they are cells that are intended to be inoculated into a culture container and cultured, especially cells that are expected to be uniformly inoculated into a culture container. They can be a single cell or a combination of two or more cells. For example, stem cells, adipocytes, hepatocytes, kidney cells, pancreatic cells, breast cells, corneal cells, endothelial cells, epithelial cells, epidermal cells, smooth muscle cells, myoblasts, cardiomyocytes, nerve cells, glial cells, dendritic cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, fibroblasts, blood cells, mesenchymal cells and their precursor cells can be listed. The cells can be normal cells or disease cells, such as tumor cells such as cancer cells. In a preferred embodiment, the cells are stem cells, such as induced pluripotent stem cells (Induced pluripotent stem cells: iPS cells), embryonic stem cells (Embryonic stem cells: ES cells), mesenchymal stem cells, etc. Stem cells can be undifferentiated stem cells (such as undifferentiated iPS cells) or cells partially or completely differentiated from stem cells. The cells may be naturally derived cells, or may be chemically or physically treated cells, genetically manipulated cells, or the like.

[0105] In addition, the source of the cell is not particularly limited, for example, it is an animal, preferably a mammalian cell, specifically, it can be a cell from a primate (human, monkey, chimpanzee, gorilla, etc.), an experimental animal (mice, rats, etc.), a livestock animal (cow, pig, rabbit, etc.), a pet animal (dog, cat, etc.). When the preparation of a cell preparation is targeted, for example, cells from a human patient can be used as autologous cells. In addition, when the preparation of a disease model cell is targeted, for example, cells of the disease site isolated from a patient suffering from the disease can be used, or a known cell line of the disease, a cell into which a pathogenic gene of the disease has been introduced, etc. can be used.

[0106] Cell can be prepared by methods well known in the art, for example, can be cultured in appropriate culture medium. The kind of culture medium is not particularly limited, for example, any cell culture minimal medium, differentiation medium, primary culture special culture medium etc. can be used. Specifically, Dulbecco's modified Eagle's medium (DMEM), StemFit culture medium, Glasgow's MEM (GMEM), RPMI1640, Ham's F12, MCDB culture medium, Williams culture medium E etc. can be listed, but are not limited to these, as long as the culture medium containing the proliferation of cells, the component required for differentiation then all can be utilized. And then, the culture medium with the addition of serum, various growth factors, differentiation-inducing factors can be used.

[0107] In this specification, "dispenser" refers to a machine having a mechanism for aspirating and discharging a cell suspension (e.g., a pipette), which can be manual or automatic. The dispensing machine is preferably an automated dispensing machine. In one embodiment, the automated dispensing machine can be an automated dispensing machine that can control the aspiration and discharge steps with high precision (e.g., a dispensing machine operated by LabDroid Maholo).

[0108] First, prepare a cell suspension. A cell suspension refers to a liquid obtained by suspending cells in a liquid medium. The form of the cells in the cell suspension can be the form of dispersed single cells or the form of a cell mass comprising multiple cells. The liquid medium is different depending on the type of cells used, and a liquid medium suitable for the cell culture performed thereafter can be selected. For example, Dulbecco's modified Eagle's medium (DMEM), StemFit culture medium, Glasgow'sMEM (GMEM), RPMI1640, Ham's F12, MCDB culture medium, Williams culture medium E, etc. can be listed.

[0109] The concentration of the cell suspension can be appropriately set by those skilled in the art in consideration of the type of cells, the amount of liquid discharged, the presence or absence of culture medium pre-added to the culture vessel, the cell density after seeding the cell suspension in the culture vessel, etc. The cell density after seeding the cell suspension varies depending on the type of cells and the purpose of cell culture, and can be set, for example, to 10 2 ~10 6 cells / cm 2 , 10 2 ~10 5 cells / cm 2 , 10 3 ~10 6 cells / cm 2 , preferably set to 10 3 ~10 5 cells / cm 2 For example, when differentiation induction is performed after culture, it is also conceivable that the cell density in the culture vessel may be set higher or lower than that in simple cell culture.

[0110] In this cell seeding method, a cell suspension is aspirated using a dispenser. The amount of cell suspension aspirated can be determined based on the total amount of cell suspension to be discharged into the culture vessel and the number of discharges, but is limited by the maximum aspiration capacity of the dispenser. For example, when a total of 5 to 15 mL of cell suspension is to be discharged in 1 to 4 times, 1.25 to 15 mL of cell suspension is aspirated each time. When the aspiration step is performed two or more times, the amount of cell suspension aspirated can be the same or different each time. The aspiration speed is not particularly limited as long as it is a conventional speed.

[0111] Then, the cell suspension is discharged to a fixed point in the culture container using a dispenser. In this specification, "culture container" refers to a container in which cells are inoculated by this cell inoculation method and the cells inoculated therein are cultured. Culture containers are well known in the art, and culture containers of various forms and shapes are available for sale. Those skilled in the art can appropriately select the shape, material and size (culture area) of the culture container according to the type of cells used, the form and purpose of culture, the scale of culture, etc. As examples of culture containers, holes in culture plates such as single-hole plates or multi-hole plates, specifically holes in 1-hole plates, each hole in 6-hole plates, each hole in 12-hole plates, etc., culture plates, culture dishes, etc. can be listed. In the case of a multi-hole plate or the like having multiple holes in 1 plate, each hole is regarded as a culture container in this specification. In one embodiment, the culture container can be a hole in 1-hole plate, 1 hole in 6-hole plates, or 1 hole in 12-hole plates. In addition, the shape of the culture container is not limited, and can be circular, elliptical, quadrilateral (square, rectangle), etc., and flat-bottomed or round-bottomed. When it is desired that the cell density of stem cells or the like is uniform, a flat-bottomed culture container is preferably used. In one embodiment, the culture container is a hole in a quadrilateral flat-bottomed 1-well plate. The material of the culture container can be any material commonly used in cell culture, and examples thereof include: quartz; inorganic glass such as glass and borosilicate glass; carbon; metals such as gold, silicon, nickel, titanium, and aluminum; polyolefins such as polyethylene and polypropylene; polyesters such as polybutylene terephthalate (PBT) and polyethylene terephthalate (PET); cyclic olefin resins; acrylic resins such as polymethyl methacrylate (PMMA); epoxy resins, etc., but are not limited thereto. The size (culture area) of the culture container is not particularly limited, and for example, 3 cm 2 Above, for example 9cm 2 Above, 90cm 2 Above, 3-500cm 2 3~200cm 2 3~100cm 2 , 9~500cm 2 , 9~200cm 2 , 9~100cm 2 20~500cm 2 20~200cm 2 20~100cm 2 40~500cm 2 40~200cm 2 40~100cm 2 , 60~500cm 2 , 60~200cm 2 , 60~150cm 2 , 60~140cm 2, 60~120cm 2 , 60~100cm 2 70~500cm 2 70~200cm 2 70~150cm 2 70~140cm 2 70~120cm 2 70~100cm 2 80~500cm 2 , 80~200cm 2 , 80~150cm 2 , 80~140cm 2 , 80~100cm 2 90~500cm 2 90~200cm 2 90~150cm 2 90~140cm 2 90~100cm 2 or 95cm 2 In one embodiment, the culture container has a culture area of ​​3 to 100 cm 2 In one embodiment, the culture container has a culture area of ​​60 to 150 cm 2 (e.g. 60-120cm 2 , 60~100cm 2 70~150cm 2 70~120cm 2 , or 70-100cm 2 In one embodiment, the culture container has a culture area of ​​90 to 100 cm 2 In one embodiment, the culture container is a well of a well plate or a well of a multi-well plate and has a volume of 3 to 100 cm 2 In one embodiment, the culture container is a well having a culture area of ​​60 to 150 cm 2 (e.g. 60-120cm 2 , 60~100cm 2 70~150cm 2 70~120cm 2 or 70-100cm 2 ) of a well of a well plate. In one embodiment, the culture container is a well having a culture area of ​​90 to 100 cm 2 The culture area of ​​a well of a 1-well plate.

[0112] In one embodiment, a culture fluid may be added to the culture vessel in advance. For example, based on the concentration and output of the cell suspension, the culture fluid may be added in advance in a manner such that the cell density (cell density in the culture vessel) suitable for cell culture is achieved, thereby adjusting the final cell density. The culture fluid may vary depending on the type of cells used and the purpose of cell culture, and those skilled in the art may appropriately select the culture fluid. The culture fluid may be the same as or different from the culture fluid used in the cell suspension.

[0113] In one embodiment, the culture vessel may be partially or completely coated with a coating agent (e.g., a culture matrix). Alternatively, a coating agent may be added to the culture solution pre-added to the culture vessel. The coating agent is not particularly limited as long as it is a coating agent commonly used for cell culture. For example, as a coating agent for iPS cells, iMatrix-511 (manufactured by Nippi), Matrigel (Matrigel) (registered trademark), vitronectin, etc. may be mentioned. In another embodiment, feeder cells may be cultured in the culture vessel. Operations commonly performed in cell culture may also be appropriately performed in the present invention.

[0114] "Discharging to a fixed point" means discharging the cell culture fluid to a single point within the culture vessel. When the discharging step is performed two or more times, the discharge is performed at exactly or substantially the same point. This single point is ideally located on the bottom surface of the culture vessel, particularly at the center of the bottom surface. The center refers to the center or approximately the center.

[0115] The amount of cell suspension discharged and the number of discharges are determined based on the total amount of cell suspension discharged into the culture vessel. As described above, these are roughly equivalent to the amount and number of times the cell suspension is aspirated. It is believed that if the amount discharged per time is too small or the number of discharges is too large, cells will tend to be concentrated toward the center of the culture vessel. Therefore, the amount and number of discharges should be determined based on factors such as the cell type, the concentration of the cell suspension, and the type of liquid medium.

[0116] The speed of discharging the cell suspension is a factor that affects the uniform inoculation of cells into the culture container. The appropriate speed can be selected by considering the cell concentration and viscosity of the cell suspension, the amount of cell suspension discharged, the height of the cell suspension discharged, etc. In one embodiment, as the discharge speed, 350 to 1350 μL cell suspension / second, preferably 850 to 1100 μL cell suspension / second can be used. For example, by performing the experiment described in Example 4, the discharge speed suitable for the cell suspension used can be obtained. As an example, the cells are discharged at a rate of 10 2 ~10 7 cells / mL, preferably 10 4 ~10 6When a cell suspension is suspended in a culture medium at a concentration of 10 cells / mL and discharged three times, each time in an amount of about 5 mL, the discharge rate can be set to 350 to 1350 μL / second, preferably 850 to 1100 μL / second. It should be noted that in Example 4, iPS cells were used to study the conditions, but the type of cells is not particularly limited, and the same discharge rate can be used. The discharge rate can be fixed or varied within a certain range (for example, the discharge rate can be changed during a single discharge, or the discharge rate can be different during each discharge).

[0117] The height when discharging the cell suspension (the distance from the bottom surface of the culture vessel to the discharge point at the front end of the dispenser) is the height at which the front end of the dispenser does not touch the bottom surface of the culture vessel. In order to avoid the influence of bubbles generated during discharge and cause the cell density to become uneven, it is preferably more than 10 mm from the bottom surface of the culture vessel. As long as the height is below the degree that the cell suspension or culture solution does not scatter during discharge (for example, about 5 cm), it can be allowed. In one embodiment, the discharge height is a height at which the dispenser does not insert into the interior of the culture vessel, for example, the same height as the wall of the culture vessel or higher. In another embodiment, the discharge height is a height of more than 10 mm from the bottom surface of the culture vessel, but the discharge is performed in a state where the dispenser is inserted into the interior of the culture vessel.

[0118] In one embodiment, the cell suspension is not sprayed or discharged in the form of droplets.

[0119] In the present cell seeding method, the aspiration step and the discharge step can be performed once or twice or more, for example, 2 to 4 times. The number of times is set according to the total amount of cell suspension discharged and the discharge volume per time.

[0120] In one embodiment, the cell seeding method further comprises the step of allowing the cells to adhere to the culture container by allowing the culture container to stand without shaking after the final discharge step. The standing time can be appropriately set depending on the cell type and the treatment of the culture medium. For example, the standing time can be 1 to 30 minutes, preferably 5 to 20 minutes.

[0121] In this specification, "oscillation" refers to vibrating the culture container in a substantially horizontal direction. Common oscillations used in the field of cell culture include reciprocating oscillation, rotational oscillation, and figure-8 oscillation. Those skilled in the art can appropriately select the type of oscillation according to the purpose.

[0122] In this cell seeding method, the culture vessel is preferably not shaken after the discharge step, but it is conceivable that shaking may be necessary, for example, when the viscosity of the cell suspension is high. It should be noted that this cell seeding method does not include the stirring operation after the discharge step that is sometimes performed to uniformly distribute the cells (for example, stirring achieved by repeatedly aspirating and discharging the culture vessel using a dispenser).

[0123] In one embodiment, the present cell seeding method further comprises a step of stirring the cell suspension prior to the aspiration step. This stirring step is particularly preferred when using cells that tend to settle in the cell suspension. The cell suspension can be stirred using commonly used stirring methods in the cell culture field, for example, by repeated aspiration and discharge operations using a dispenser, or by oscillation.

[0124] In a preferred embodiment, the present cell inoculation method further includes a step of stirring the cell suspension before the aspiration step, and then the aspiration step and the discharge step are performed once or twice or more. It should be noted that when the aspiration step and the discharge step are performed twice or more, it is preferred to implement the stirring step before the first aspiration step and the discharge step. However, when cells with a fast cell sedimentation rate are used, stirring may be performed twice or more before each aspiration step and the discharge step. In one embodiment, after the stirring step, the aspiration step and the discharge step are completed before the cells in the cell suspension settle. In addition, when multiple culture containers, such as multi-well plates, are used, after the step of stirring the cell suspension, the aspiration step and the discharge step may be continued for each culture container (or a portion of the culture container). For example, after the step of stirring the cell suspension, the aspiration step and the discharge step are performed for 2 to 12 culture containers, such as 2 to 6 culture containers. Alternatively, the stirring step may be performed separately before the aspiration step and the discharge step are performed for each culture container (the stirring step, the aspiration step and the discharge step are performed for each culture container).

[0125] The amount of the prepared cell suspension can be appropriately set by those skilled in the art in consideration of the total amount of the cell suspension to be discharged into the culture container and an appropriate amount for uniformly stirring the cells in the stirring step before the aspiration step.

[0126] In one embodiment, the cell seeding method is a method of cell seeding using an automated dispenser, comprising:

[0127] Steps for aspirating cell suspension using a dispenser;

[0128] a step of discharging the cell suspension to a fixed point in the culture container using the dispenser; and

[0129] After the discharge step, the culture container is left to stand without shaking to allow the cells to adhere to the culture container.

[0130] In one embodiment, the cell seeding method is a method of cell seeding using an automated dispenser, comprising:

[0131] Steps for aspirating cell suspension using a dispenser;

[0132] a step of discharging the cell suspension to a fixed point in the culture container using the dispenser; and

[0133] After the discharge step, the culture container is left to stand without shaking to allow the cells to adhere to the culture container.

[0134] The fixed point in the culture container is located at the center of the bottom surface of the culture container.

[0135] The culture container is a culture container with a flat bottom (for example, a quadrilateral flat bottom).

[0136] In one embodiment, the cell seeding method is a method of cell seeding using an automated dispenser, comprising:

[0137] Steps for aspirating cell suspension using a dispenser;

[0138] a step of discharging the cell suspension to a fixed point in the culture container using the dispenser; and

[0139] After the discharge step, the culture container is left to stand without shaking to allow the cells to adhere to the culture container.

[0140] The fixed point in the culture container is located at the center of the bottom surface of the culture container.

[0141] The above-mentioned culture container is a culture container with a flat bottom (for example, a quadrilateral flat bottom).

[0142] The above cells are stem cells, preferably iPS cells.

[0143] In one embodiment, the cell seeding method is a method of cell seeding using an automated dispenser, comprising:

[0144] Steps for aspirating cell suspension using a dispenser;

[0145] a step of discharging the cell suspension to a fixed point in the culture container using the dispenser; and

[0146] After the discharge step, the culture container is left to stand without shaking to allow the cells to adhere to the culture container.

[0147] The fixed point in the culture container is located at the center of the bottom surface of the culture container.

[0148] The culture container is a flat bottom (e.g., a quadrilateral flat bottom) with a volume of 3 to 500 cm. 2 (For example, 3 to 200 cm 2 3~100cm 2 , 9~500cm 2 , 9~200cm 2 , 9~100cm 2 20~500cm 2 20~200cm 2 20~100cm 2 40~500cm 2 40~200cm 2 40~100cm 2 , 60~500cm 2 , 60~200cm 2 , 60~150cm 2 , 60~140cm 2 , 60~120cm 2 , 60~100cm 2 70~500cm 2 70~200cm 2 70~150cm 2 70~140cm 2 70~120cm 2 70~100cm 2 80~500cm 2 , 80~200cm 2 , 80~150cm 2 , 80~140cm 2 , 80~100cm 2 90~500cm 2 90~200cm 2 90~150cm 2 90~140cm 2 90~100cm 2 or 95cm 2 ) of a culture container with a culture area (e.g., a well of a 1-well plate),

[0149] The above cells are stem cells, preferably iPS cells.

[0150] In one embodiment, the cell seeding method is a method of cell seeding using an automated dispenser, comprising:

[0151] Steps for aspirating cell suspension using a dispenser;

[0152] a step of discharging the cell suspension to a fixed point in the culture container using the dispenser; and

[0153] After the discharge step, the culture container is left to stand without shaking to allow the cells to adhere to the culture container.

[0154] The fixed point in the culture container is located at the center of the bottom surface of the culture container.

[0155] The above-mentioned culture container is a quadrilateral with a flat bottom and a 60-150 cm 2 (e.g. 60-120cm 2 , 60~100cm 2 70~150cm 2 70~120cm 2 or 70-100cm 2 ) of the well of a 1-well plate with a culture area of

[0156] The above cells are iPS cells.

[0157] (Cell seeding device)

[0158] In one embodiment, the present invention provides a cell seeding device comprising a dispenser, a portion for holding a cell suspension, a portion for holding a culture container, and a control unit.

[0159] The control unit is controlled in the following manner:

[0160] The dispensing machine is caused to aspirate the cell suspension held by the cell suspension holding portion.

[0161] The cell suspension sucked into the dispenser is discharged to a fixed point in the culture container held by the culture container holding portion.

[0162] In order to ensure that the cell is discharged from the dispenser to a fixed point in the culture container, the cell seeding device may include a mechanism for aligning the position of the center portion of the culture container in the portion holding the culture container.

[0163] The control unit may control the cell suspension held by the cell suspension holding portion to stir the cell suspension, or, for example, may control the dispenser to perform suction and discharge operations in the cell suspension held by the cell suspension holding portion.

[0164] In order to add additional components and / or liquid to the cell suspension held by the cell suspension holding portion and / or the culture container held by the culture container holding portion, the cell seeding device may include a portion for holding such additional components and / or liquid.

[0165] Furthermore, the cell seeding apparatus may include a device for observing the culture vessel held by the culture vessel holding portion (eg, a light source, a detection optical system, an image acquisition unit, an image analysis unit, etc.).

[0166] The control unit controls at least one of the following: aspiration and discharge of the cell suspension by the dispenser (including the speed and frequency of discharge, and discharge to a single point in the culture vessel), alignment of the culture vessel, stirring of the cell suspension, oscillation of the culture vessel, and addition of additional components and / or liquid to the cell suspension and / or the culture vessel. For example, the control unit is configured to control the dispenser to discharge the cell suspension to a single, fixed point in the culture vessel under appropriate discharge conditions (discharge volume, discharge rate, etc.) based on input discharge conditions or input information such as cell type and cell suspension concentration. The control unit can utilize any control means known in the art, and can be, for example, a computer.

[0167] As described above, using the method and apparatus for cell seeding of the present invention, cells can be uniformly seeded into a culture container.

[0168] (Procedure for cell seeding)

[0169] In one embodiment, the present invention provides a program for cell seeding using a dispenser, which causes the dispenser to perform the following steps:

[0170] a step of aspirating the cell suspension held in the portion holding the cell suspension;

[0171] A step of discharging the cell suspension to a fixed point in the culture container held by a culture container holding portion.

[0172] In one embodiment, the program of the present invention can cause the dispenser to perform an aspiration step and a discharge step once or twice or more (e.g., 2 to 4 times). In one embodiment, the program of the present invention can cause the dispenser to further perform a step of stirring the cell suspension before the aspiration step. For example, the aspiration step and the discharge step are preferably completed after the stirring step and before the cells in the cell suspension settle. In one embodiment, in the program of the present invention, a fixed point in the culture container is set to the center of the bottom surface of the culture container.

[0173] About the content of the control of utilizing program, in addition to making the dispensing machine aspirate cell suspension and discharge it into the culture container, it is also possible to list making the dispensing machine stir the cell suspension, making the control unit calculate the appropriate cell discharge speed based on the type of cell, the cell concentration of the cell suspension, the size of the culture container, etc., making the observation unit observe the number, state and / or form of cells, making the output unit display the observation results, etc. At this time, the reference data (based on the conditions of appropriate cell inoculation such as the type of cell, the cell concentration of the cell suspension, the size of the culture container, etc.) pre-stored in the storage unit can also be utilized. Furthermore, the reference data can also be updated by sending and receiving information with the external network. It should be noted that the functional control of the dispensing machine, etc. can be realized by software by interpreting and executing the program stored in the memory of the control unit by the processor. The information such as the program of each function, the database, etc. can also be stored in the recording device such as memory, hard disk, SSD (Solid State Drive, solid state drive) or the recording medium such as IC card, SD card, DVD.

[0174] When the program of the present invention is installed on a computer, for example, the following steps may be performed:

[0175] (S0) Inputting information such as the type of cells to be seeded, the concentration of the cell suspension, and the size of the culture container (e.g., input by the user, selection on a selection panel, etc.);

[0176] (S1) Based on the input information, the dispensing machine stirs the cell suspension held by the portion holding the cell suspension as needed;

[0177] (S2) Based on the input information, the dispensing machine aspirates an appropriate amount of the cell suspension held by the portion holding the cell suspension;

[0178] (S3) Based on the input information, the dispensing machine discharges the cell suspension at an appropriate amount and speed to a fixed point in the culture container held by the part holding the culture container;

[0179] (S4) The number, state, and / or morphology of cells in the culture container are observed by the observation unit as needed, and the observation result is displayed by the output unit as needed.

[0180] (Method for dispensing cell suspension)

[0181] In another embodiment, the present invention provides a method for dispensing a cell suspension using a dispenser, comprising: aspirating the cell suspension using the dispenser; and discharging the cell suspension into a fixed point within a culture vessel using the dispenser. Each step can be performed as described above. In this specification, "dispensing" refers to distributing a predetermined amount of cells onto a culture vessel or into a culture medium within the culture vessel.

[0182] (Cell culture method and cell culture system)

[0183] In one embodiment, the present invention provides a method for culturing cells in a culture container, comprising:

[0184] A step of seeding cells into a culture container using the cell seeding method described in this specification; and

[0185] The step of culturing the above cells in the above culture container.

[0186] The step of culturing cells is carried out by a method suitable for the type of cells used and the purpose of culture (e.g., proliferation, differentiation, etc.). Regarding the culture conditions including culture form (e.g., static culture, shaking culture, etc.), culture temperature, pH, presence or absence of ventilation, and culture time, those skilled in the art can appropriately set them. As needed, after the cell inoculation step, culture medium, culture medium components, nutrients, etc. for culture can be added to the culture vessel, or culture medium exchange can be performed. For example, the culture step is carried out for a suitable time at 32 to 37 ° C and pH 4.0 to 8.0 under static culture or shaking culture. When the pH changes due to cell culture, an inorganic or organic acid, alkaline solution, etc. can be used to adjust the pH.

[0187] Before, during, and / or after the cell culture step, the number, state, and / or morphology of cells can be confirmed visually or by imaging, and the repetition or termination of the cell culture can be determined as needed.

[0188] The cell culture method may further include the step of transferring the culture container to an incubator. In this case, the step of culturing cells is performed in the culture container in the incubator.

[0189] The present cell culture method may further comprise the step of inducing differentiation of the cells. When the cells are precursor cells or stem cells, the cells may be differentiated into target cells by performing appropriate differentiation induction treatment. The differentiation induction treatment may be performed by adding differentiation inducing factors to the culture medium, inducing the expression of specific genes in the cells, contacting with other cells, etc., and may vary depending on the type of cells before differentiation induction and the type of cells to be differentiated into. Those skilled in the art may select appropriate differentiation induction treatment based on the technical common sense in this technical field, and a variety of kits for differentiation induction are also available on the market.

[0190] Differentiation-inducing factors are not particularly limited as long as they are commonly used in the art, and examples thereof include growth factors (vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), osteogenic factor 4, transforming growth factor-β (TGFβ) superfamily, insulin-like growth factor (IGF), epithelial cell growth factor (EGF), nerve growth factor (NGF), etc.), horse serum (less than 5%), Wnt signal activating factors (CHIR99021, etc.), Wnt signal inhibitors (IWR1, IWP2, XAV939, Dkk-1, etc.). Such differentiation-inducing factors can be used alone or in combination of two or more. Those skilled in the art can appropriately select the type of cells used and the type of target cells to be differentiated into. Alternatively, on this basis, specific gene expression in cells can be induced to induce differentiation. For example, gene expression of differentiation-inducing factors in cells can be induced.

[0191] The culture medium used for differentiation induction is not particularly limited as long as it is a basic medium commonly used in the art, and examples thereof include Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), α-modified Eagle's Minimum Essential Medium (αMEM), StemFit medium, Glasgow's MEM (GMEM), RPMI1640, Ham's F12, MCDB medium, Williams medium E, mesenchymal cell basal medium (MSCBM), E6 (Essential 6), etc. These media may be supplemented with amino acids, inorganic salts, vitamins, antibiotics, etc., as needed.

[0192] Cell culture conditions during differentiation induction can be conventional conditions for culturing animal cells. For example, the conditions can be 32-37°C, a CO2 concentration of 1-10% and an O2 concentration of 2-21% (e.g., approximately 21% (oxygen concentration in the atmosphere)), and a pH of approximately 4.0-8.0 for an appropriate period of time.

[0193] In one embodiment, cells can be induced to differentiate by one or more differentiation induction steps (e.g., cell culture in a culture medium containing one or more differentiation inducing factors). Regarding the differentiation induction step, the same differentiation induction step can be performed once or twice or different differentiation induction steps can be performed twice or more. For example, cell culture in a culture medium containing differentiation inducing factors can be performed once or twice or more in a culture medium containing the same differentiation inducing factors or twice or more in a culture medium containing different differentiation inducing factors. For example, stem cells can be cultured in a culture medium containing differentiation inducing factors and induced to differentiate into mesenchymal stem cells, ectoderm cells, mesoderm cells, endoderm cells, etc., and then cultured in a culture medium containing other differentiation inducing factors to further differentiate into target differentiated cells or their precursor cells, and the precursor cells can be further differentiated into target differentiated cells by culture in a culture medium containing other differentiation inducing factors.

[0194] When stem cells (e.g., iPS cells) are used as cells before differentiation induction, the stem cells can be differentiated into any cells, such as mesenchymal stem cells, ectoderm cells, mesoderm cells, endoderm cells, fibroblasts, myoblasts, cardiomyocytes, adipocytes, hepatocytes, kidney cells, pancreatic cells, mammary cells, corneal cells, endothelial cells, epithelial cells, epidermal cells, skeletal muscle cells, smooth muscle cells, nerve cells, glial cells, dendritic cells, bone marrow cells, chondrocytes, osteoblasts, osteoclasts, osteocytes, etc. In addition, when specific precursor cells are used, differentiation into target cells can be induced from the precursor cells.

[0195] After the differentiation induction step, conventional methods such as immunostaining can be used to confirm whether the cells have differentiated into target cells. For example, when observing with a fluorescent microscope using a fluorescently labeled antibody (fluorescent immunostaining), antibodies that recognize marker proteins specific to the differentiated cells are used as antibodies for fluorescent immunostaining. For example, in order to detect differentiated skeletal muscle cells, immunostaining using antibodies that recognize myosin heavy chain (MyHC) can be used.

[0196] The cell culture method may further include a step of recovering the cultured cells or differentiated cells from the culture vessel. Before or after the cell recovery step, the number, state, and / or morphology of the cells may be confirmed visually or by imaging.

[0197] In another embodiment, the present invention provides a cell culture system comprising the above-mentioned cell seeding device, an incubator, a transfer mechanism, and a control unit.

[0198] The control unit is controlled in the following manner:

[0199] The culture container is transferred from the culture container holding portion of the cell seeding device to the incubator by means of the transfer mechanism.

[0200] The cells in the culture container are cultured in the incubator.

[0201] The cell culture system of the present invention may include an observation unit for observing the culture container and cells in the culture container, an input unit for user input, an output unit, and the like.

[0202] (Method for preparing cell reserves)

[0203] In another embodiment, the present invention provides a method for producing a cell stock, comprising: seeding cells in a culture vessel using the cell seeding method described herein; and culturing the cells in the culture vessel.

[0204] The preparation method of this cell reserve material may further include the step of transferring the culture container to an incubator, in which case, the step of culturing cells in the culture container in the incubator. In addition, the preparation method of this cell reserve material may further include the step of inducing differentiation of the cells. Further, the preparation method of this cell reserve material may further include the step of reclaiming the cultured cells or differentiated cells from the culture container. Before or after the cell recovery step, the number, state and / or form of the cells may be confirmed visually or by image capture.

[0205] Example

[0206] While the present invention has been generally described, specific examples are provided herein for further understanding. These examples are provided for illustrative purposes only and are not intended to limit the present invention.

[0207] All cell culture and differentiation induction were carried out at 37°C, 5% CO2, and atmospheric oxygen concentration.

[0208] [Example 1] Cell seeding based on two-point discharge

[0209] The ROCK inhibitor Y-27632 (Wako, 036-24023, final concentration 10 μM) and the coating agent iMatrix-511 (Nippi, 892012, final concentration 0.25 μg / cm 2iPS cells (201B7 (Takahashi, K. et al., Cell. 30; 131(5): 861-872)) were cultured on a 6-well plate (Iwaki, 3810-006) for several days to prepare a sufficient amount of cells. The addition period of Y-27632 was set to the second day after cell inoculation. The proliferated iPS cells were recovered in a 15 mL centrifuge tube using the dissociation agent TrypLE Select (Gibco, 12563-011) and washed by centrifugation. After removing the supernatant, the cells were suspended in an appropriate amount of StemFit medium. The cell suspension was counted and the cell density at the time of cell inoculation was about 2000 cells / cm 2 The cell suspension (approximately 1.27 × 10 4 cells / mL) were added into a 50 mL centrifuge tube.

[0210] A 1-well plate (culture area 95 cm) was used as a culture container. 2 ; Greiner, 670180), 3 mL of iPS cell culture medium and Hoechst for nuclear staining (Invitrogen, H1399, final concentration 1 μg / mL) were added before cell seeding.

[0211] The above-mentioned 1-well plate is different from the circular culture dish commonly used for cell culture and has a rectangular shape suitable for automation. Therefore, it is believed that in order to uniformly seed cells, it is necessary to discharge the cell suspension from one point in the center of each of the two areas divided by a straight line bisecting the long side perpendicularly, for a total of two points.

[0212] The following cell seeding procedure was performed using the LabDroid Maholo (Maholo), which is capable of automated dispensing and cell culture in an incubator. 5 mL of the cell suspension was aspirated and discharged four times within a 50 mL centrifuge tube to uniformly stir the cell suspension. Since the Maholo's electronic pipette (Viaflo II; Integra, #4015) delivers less than 5 mL of liquid per single discharge, cells were seeded into a single-well plate under the following conditions.

[0213] (i) To inoculate 15 mL / well of the cell suspension, 3.75 mL of the cell suspension was dispensed twice each at two points, for a total of four times.

[0214] (ii) 5 mL of cell suspension was discharged to two points respectively, twice in total. In order to make the volume of the culture container after inoculation reach 18 mL, the volume of iPS cell culture medium added to the culture container was set to 8 mL only at this time. In addition, in order to make the cell density at the time of cell inoculation the same as in (i), the iPS cell suspension was appropriately diluted to 1.90 × 10 4 A cell suspension of 10 cells / mL was added to a 50 mL centrifuge tube and inoculated from the 50 mL centrifuge tube. Under both conditions (i) and (ii), the cell suspension was discharged from a height of +17 mm from the bottom of the culture vessel at a rate of 890 μL / s.

[0215] After the cells were seeded, they were allowed to stand for 10 minutes, and after the cells adhered to the culture container, they were placed in an incubator for cell culture (37°C, 5% CO2). The doubling time of the iPS cells used was about 30 hours, so it was considered that within 10 hours from the start of culture, the iPS cells proliferated before proliferation and did not affect the evaluation results. Therefore, the uniformity evaluation of the cell seeding density was implemented within 10 hours from the start of culture. The confocal imaging analysis system CQ1 (Yokogawa Electric) was used to shoot the entire surface of the culture container (a total of 240 fields of view). Using the analysis software CellPathfinder (Yokogawa Electric), after measuring the number of Hoechst-positive particles in each field of view (counts / field), the CV value and maximum / average value as indicators of the uniformity of the cell seeding density were calculated. When calculating the CV value and the maximum / average value, the measured values ​​of 180 fields of view other than the outermost field of view of the culture container were used out of the 240 fields of view shot. This is because the insufficient brightness of the edge of the hole in the visual field and the inner wall of the hole will affect the accurate identification of the cells when reflecting light. The CV value is a value for evaluating the deviation of the number of cells in the entire hole, usually obtained by standard deviation (SD) / mean value, and the smaller the value, the smaller the deviation. The maximum / mean value is the value obtained by dividing the maximum value (Max) of the count value / field of view by the mean value (Mean), which is a value for evaluating how much higher the cell density is than the assumption when the cell density locally becomes higher. The preferred maximum / mean value is a small way. About the uniformity of the cells in the culture container, in addition to the CV value and the maximum / mean value, it can also be judged by the heat map made based on the number of cells in each visual field. The heat map is a graph showing the measured values ​​of each visual field in the above-mentioned 180 visual fields with color depth. The smaller the difference in the color depth of the heat map based on visual observation, the more uniform it is, and it can be judged that there is no deviation in local cell density.

[0216] The results are shown in Tables 1 and Figures 1-2 . Figure 1 Show the result of condition (i), Figure 2The result of condition (ii) is shown. Figure 1 and 2 In the figure, A shows the cell density (number of cells per field of view), and B shows a heat map based on the cell density.

[0217] [Table 1]

[0218]

[0219] In both cases (i) and (ii), cells were distributed more around two points, indicating that the properties of cells may have become heterogeneous during cell inoculation and differentiation induction (Tables 1 and Figures 1-2 ).

[0220] [Example 2] Study on Cell Seeding Based on One-Point Discharge and the Oscillation of Culture Containers

[0221] In Example 1, we found that cells were highly distributed around the two discharge points. Therefore, we decided to fix the discharge point of the cell suspension at a single point in the center of the culture vessel. Furthermore, since we expected that shaking would uniformly spread the cells, we investigated whether or not to shake the culture vessel after discharge.

[0222] The method described in Example 1 was used to prepare iPS cells and then suspend the cell suspension using a Maholo. Next, 5 mL of the cell suspension was aspirated using the Maholo and discharged into a single point in the center of the culture vessel. This discharge operation was repeated three times, until 15 mL of the cell suspension was discharged into the culture vessel. The discharge height and discharge speed were identical to those in Example 1. Cells were seeded into three culture vessels using the same conditions.

[0223] Immediately after discharging the cell suspension, two of the three culture containers were moved horizontally along the left and right directions for a certain distance (amplitude is about 10 cm or less) at a speed of 90 mm / second, and then returned to their original positions. Then, they were moved horizontally along the front and back directions for a certain distance (amplitude is about 10 cm or less) and returned to their original positions. The above-mentioned action was used as an oscillation, and the culture containers were oscillated 10 or 20 times. It should be noted that, in the present embodiment, the left and right directions of oscillation refer to the direction of the axis along the short side parallel to the well plate, and the front and back directions refer to the direction of the axis along the long side parallel to the well plate. The three culture containers were left to stand for 10 minutes, and after the cells adhered to the culture containers, they were placed in an incubator (37°C, 5% CO2) for cell culture. Within 10 hours from the start of culture, the uniformity of the cell seeding density was evaluated by the same method as in Example 1.

[0224] The results are shown in Table 2 and Figures 3-5 . Figure 3 The results of 10 oscillations are shown. Figure 4 The results of 20 oscillations are shown. Figure 5 Results without oscillations are shown. Figures 3-5 In the figure, A shows the cell density (number of cells per field of view), and B shows a heat map based on the cell density.

[0225] [Table 2]

[0226]

[0227] The results show that the method of discharging at one point can seed cells more evenly than that of discharging at two points. In addition, the method of not shaking the culture container after discharging the cell suspension can seed cells evenly (Table 2 and Figures 3-5 ).

[0228] [Example 3] Study on stirring method

[0229] In order to inoculate cells more evenly, the stirring method using Maholo to stir the cell suspension was studied. In Example 2, after the cell suspension was stirred 4 times in a 50mL centrifuge tube, 5mL of the cell suspension was transferred 3 times (suction and discharge) from the centrifuge tube to the culture vessel, thus inoculating cells. A part of this action was changed. In the present embodiment, after stirring 4 times in a 50mL centrifuge tube, 5mL of the cell suspension was transferred once from the centrifuge tube to the culture vessel, and this action was implemented for 3 cycles, thus inoculating cells (stirring every time). Except the stirring of the cell suspension and the conditions of the transfer, all were implemented according to the same conditions as in Example 2 without oscillation.

[0230] The results are shown in Figure 6 . Figure 6 In the figure, A shows the cell density (number of cells per field of view), and B shows a heat map based on the cell density. Furthermore, under the condition of stirring each time, the CV value was 19%, and the maximum / average value was 150%. Under the condition of stirring each time, the distribution of cells was acceptably uniform, but slightly biased toward the center of the culture vessel ( Figure 6 ). Therefore, it can be seen that stirring can be performed each time the cell suspension is discharged into the culture container, but the method of continuously performing the transfer (suction and discharge) operation after stirring once as in Example 2 can more uniformly seed the cells.

[0231] [Example 4] Study on the discharge rate of cell suspension

[0232] As shown in Example 2, for the purpose of uniformly seeding cells, the condition of not shaking the culture vessel after draining the cell suspension is optimal. It is speculated that the distribution of cells centered at a central point depends on the discharge speed of the cell suspension, with lower speeds resulting in a greater concentration in the center and higher speeds resulting in a greater distribution toward the edges. Therefore, the speed at which the cell suspension is drained into the culture vessel using a Maholo was studied. The studied discharge speeds are shown in Table 3. Aside from the discharge speed, all other conditions were the same as those in Example 2, without shaking.

[0233] [Table 3]

[0234]

[0235] The results are shown in Figure 5 and Figures 7 to 14 . Figure 5 and Figures 7-13 In FIG, A shows the cell density (number of cells per field of view) at the indicated discharge speeds, and B shows a heat map based on the cell density. Figure 14 A and B represent the CV value and the maximum value / average value at the indicated discharge rate, respectively. This result indicates that cells can be uniformly seeded when the discharge rate is in the range of 358 to 1315 μL / s, and in particular, cells can be more uniformly seeded when the discharge rate is in the range of 890 to 1078 μL / s ( Figure 5 and Figures 7 to 14 ). Therefore, it can be seen that the condition without shaking in Example 2 is the optimal condition for uniformly seeding cells.

[0236] [Example 5] Study on the reproducibility of cell seeding

[0237] Maholo was used to conduct an experiment to confirm the reproducibility of Example 1 and Example 2. The conditions and the obtained results are summarized in the following Table 4. Figures 15-17 . Figure 15 The results for the condition of one point discharge and no oscillation are shown. Figure 16 The results for the conditions of two point discharge and no oscillation are shown, Figure 17 The results are shown under the conditions of one-point discharge and 10 oscillation times. Figures 15-17 In the figure, A shows the cell density (number of cells per field of view), and B shows a heat map based on the cell density.

[0238] [Table 4]

[0239]

[0240] From these results, it was confirmed that the method of discharging the cell culture medium to a single point in the center of the culture container allowed for more uniform cell seeding than the method of dividing the culture container into two areas and discharging the cell culture medium to a single point in the center of each area (two points in total). In addition, the method of not having the step of shaking the culture container after discharging the cell culture medium was a condition that allowed for more uniform cell seeding (Table 4 and Figures 15-17 ).

[0241] [Example 6] Study on cell seeding conditions using HEK293T cells

[0242] HEK293T (Thermo Scientific, HCL4517), an immortalized cell line, was used instead of iPS cells. Maholo was used to verify whether the cells could be uniformly seeded into the culture vessel using the optimal conditions described in the previous examples. The culture medium used was DMEM (Sigma-Aldrich, D5796-500ML) containing 10% FBS (HyClone, SH30070.03) and 1% PenStrep (Gibco, 15070-063). The culture vessel was pre-treated with a poly-D-lysine solution (Gibco, A3890401, final concentration 4.2 μg / cm 2 ) coated 1-well plate.

[0243] The results are shown in Figure 18 . Figure 18 In the figure, A shows the cell density (number of cells per field of view), and B shows a heat map based on the cell density. In addition, the CV value is 14%, and the maximum value / average value is 124%. It is found that in the inoculation of HEK293T cells, cells can be uniformly inoculated under the same conditions as in Example 2 without shaking ( Figure 18 ).

[0244] [Example 7] Study on the homogeneity of cells after differentiation induction using iPS cells

[0245] We investigated whether it is possible to obtain highly homogeneous differentiated cells by seeding cells to a single point in the center of a culture vessel. In this example, we used the iPS cell line B7-MYOD-NMJ (Lin, CY. et al., JCI Insight. 2019; 4(18):e124299, hereinafter referred to as "201B7"), which is an iPS cell line (201B7) in which a doxycycline (Dox)-inducible MYOD expression vector was introduced. MYOD ”). MYOD is known to be the dominant gene that promotes skeletal muscle differentiation induction (Davis, RL.Et al., Cell. 1987; 51(6):987-1000). MYODWhen inducing differentiation, Dox was added to the culture medium to promote the differentiation of 201B7 MYOD Forced expression of MYOD in iPS cells can induce skeletal muscle cells to differentiate from iPS cells in a short period of time.

[0246] The cells were cultured in mTeSR1 medium (Stemcell technologies, ST-85850) supplemented with Rock inhibitor Y-27632 (Nakali Tesku, 18188-04, final concentration 10 μM) on a substrate covered with Matrigel (Corning, 354230, final concentration 10 μg / cm 2 )-coated culture vessels for several days to prepare a sufficient amount of cells. Y-27632 was added until the day after cell seeding.

[0247] Use the proliferated 201B7 MYOD Differentiation induction was performed. Regarding the inoculation conditions during differentiation induction culture, a comparison was performed between the optimal conditions of the previous embodiment, one-point discharge and no oscillation condition (hereinafter referred to as "one-point discharge condition") and two-point discharge and no oscillation condition (hereinafter referred to as "two-point discharge condition"). The culture medium used during inoculation was Primate ES cell culture medium (Reprocell, RCHEMD001), and the culture container was filled with matrigel (final concentration 10 μg / cm 2 ) coated 1-well plate pre-added with Primate ES cell culture medium containing Y-27632 (final concentration 10 μM) and used. The cell density at the time of cell seeding was approximately 2.88×10 4 cells / cm 2 (Under the condition of one point discharge, the cell suspension was adjusted to about 1.82×10 5 cells / mL, and the cell suspension was adjusted to approximately 2.74×10 5 Cells were seeded according to Table 5. After the cells were seeded, they were allowed to stand for 10 minutes to allow the cells to adhere to the culture container and then placed in an incubator for cell culture (37°C, 5% CO2). The day after seeding, the culture medium was exchanged for Primate ES cell culture medium containing 1μg / mL Dox (LKT Labs, D5897). The next day, the culture medium was exchanged for differentiation induction medium, and the cells were cultured for 7 days from the time of seeding. The differentiation induction medium used αMEM (Gibco, 12571063) containing 10% KnockOut serum replacement (Gibco, 10828028), 0.5% PenStrep, 1μg / mL Dox and 200μM 2-mercaptoethanol (Gibco, 21985023).

[0248] The entire surface of the culture container was photographed, and uniformity evaluation and homogeneity evaluation after differentiation induction were performed. When photographing, the entire surface of the culture container was photographed at a magnification of 2 times (a total of 240 fields of view) in Examples 1 to 6, but in this example, in order to further improve the analysis accuracy, it was photographed at a magnification of 4 times (851 fields of view) to obtain a clearer image. The analysis was performed using the measured values ​​of 735 fields of view, excluding the outermost field of view of the culture container, out of the 851 fields of view obtained by photographing the entire surface of the culture container. In addition to the uniformity evaluation of the inoculation density using Hoechst staining after inoculation, immunostaining, photography, and analysis of myosin heavy chain (MyHC), which becomes an indicator of skeletal muscle differentiation after differentiation induction culture, were also performed. In this example, the number of cells was very large, so there were many adjacent cell groups from the beginning after inoculation, making it difficult to accurately detect the number of Hoechst particles. Therefore, the Hoechst-positive area was used for the uniformity evaluation after inoculation. For the homogeneity evaluation of differentiated cells, the value obtained by dividing the MyHC-positive area by the Hoechst-positive area was used. For immunostaining, cells were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd., 163-20145) and then stained with an anti-MyHC antibody (eBioscience, 14-6503-82).

[0249] The uniformity evaluation at the time of inoculation and the homogeneity evaluation after differentiation induction are summarized in the following Table 5. Figures 19A to 22B . Figure 19A and 19B The cell density at the time of inoculation under one point exclusion condition is shown. Figure 20A and 20B The cell density at the time of inoculation under the conditions of two point exclusions is shown. Figure 21A and 21B The MyHC expression level per cell after differentiation induction under the conditions of one dot exclusion is shown. Figure 22A and 22B The MyHC expression level per cell after differentiation induction under the conditions of two point outs is shown.

[0250] [Table 5]

[0251]

[0252] Regarding the uniformity during inoculation, the same results as in Examples 1 to 6 were obtained, and it was confirmed that the uniformity of the conditions for one-point discharge was high (Table 5, Figure 19A and B. Figure 20Aand B). It was also found that under conditions in which the cell density at the time of inoculation was higher than that of Examples 1 to 6, cells could be uniformly inoculated under conditions of one-point discharge. Regarding the homogeneity after differentiation induction, the darker the color of the heat map, the larger the MyHC-positive area was relative to the number of cells, and it was considered that the site had undergone skeletal muscle differentiation compared to the surrounding area. On the other hand, the lighter the color, the smaller the MyHC-positive area was relative to the number of cells, and it was considered that the site had not undergone skeletal muscle differentiation or had not formed skeletal muscle compared to the surrounding area. Therefore, it can be understood that the smaller the difference in the color depth of the heat map, the more homogeneous the differentiated cells obtained. Under the conditions of two-point discharge, a particularly large difference in the color depth of the heat map was observed near the side (edge) of the culture container, and the CV value was 25% ( Figure 22A On the other hand, the CV value under the condition of one-point exclusion was 13%, indicating that highly homogeneous differentiated cells were obtained by using a highly uniform seeding method ( Figure 21A and B).

[0253] [Example 8] Study on the homogeneity of cells after differentiation induction using C2C12 myoblasts

[0254] In this example, it was investigated whether it is possible to obtain differentiated cells with high homogeneity similar to iPS cells using C2C12 (ATCC CRL-1772) cells that have the ability to differentiate into skeletal muscle.

[0255] Regarding the seeding conditions, a comparison between the one-point discharge condition and the two-point discharge condition was carried out in the same manner as in Example 7. The culture medium for seeding was DMEM containing 10% FBS (HyClone, SH30084.03) and 1% PenStrep, and the culture container was iMatrix-511 (final concentration 0.25 μg / cm 2 ) was coated on a 1-well plate. The cell density at the time of cell seeding was set at approximately 3×10 4 cells / cm 2 (Under the condition of one point discharge, the cell suspension was adjusted to about 1.90×10 5 cells / mL, and the cell suspension was adjusted to approximately 2.85×10 5cells / mL), and the cells were inoculated according to Table 6. On the second day of inoculation, the culture medium was replaced with a differentiation induction medium, and differentiation induction was carried out for about 3 days. The differentiation induction medium used DMEM containing 2% horse serum (Gibco, 16050-122) and 1% PenStrep. During immunostaining, after fixation with 4% paraformaldehyde, anti-MyHC antibody (Invitrogen, 50-6503-82) was used for staining. In addition, regarding the evaluation of uniformity just after inoculation, since single cell detection can be performed, the number of Hoechst particles was measured in the same manner as in Examples 1 to 6. Regarding the evaluation of homogeneity after differentiation, the value obtained by dividing the MyHC-positive area by the Hoechst-positive area was used in the same manner as in Example 7.

[0256] The uniformity evaluation at the time of seeding and the homogeneity evaluation after differentiation induction are shown in Figures 23A to 26B . Figure 23A and 23B The cell density at the time of inoculation under one point exclusion condition is shown. Figure 24A and 24B The cell density at the time of inoculation under the conditions of two point exclusions is shown. Figure 25A and 25B The MyHC expression level per cell after differentiation induction under the conditions of one dot exclusion is shown. Figure 26A and 26B The MyHC expression level per cell after differentiation induction under the conditions of two point outs is shown.

[0257] [Table 6]

[0258]

[0259] In the inoculation of C2C12 myoblasts, cells can be uniformly inoculated by evacuating at one point. In addition, it is known that cells can be uniformly inoculated under conditions where the cell density at the time of inoculation is high compared to the conditions of Examples 1 to 6 (Table 6, Figure 23 and Figure 24). Regarding the homogeneity after differentiation induction, it is believed that, as in Example 7, the darker the color depth of the heat map is, the more skeletal muscle differentiation has been performed compared to the surrounding area, and the lighter the color depth is, the more homogeneous the differentiated cells are obtained. Under the condition of two-point discharge, the difference in the color depth of the heat map can be observed in the comparison of the left and right long sides of the culture container. Moreover, the difference in the color depth of the heat map was also observed at the corner 4 of the culture container, and the CV value was 21% (Figure 26). On the other hand, the CV value of the condition of one-point discharge is 13%. This embodiment also shows that differentiated cells with high homogeneity are obtained by using an inoculation method with high uniformity (Figure 25).

[0260] Industrial applicability

[0261] By using the method of the present invention, the cell density on the culture container can be controlled without local deviation, and homogeneous cells can be mass-produced with good reproducibility. In addition, when the cultured cells are subjected to differentiation induction, homogeneous differentiated cells can also be mass-produced with good reproducibility. It is expected that the present invention will be useful in fields such as cell culture, drug development, and the manufacture of clinical cell preparations.

Claims

1. A method for cell seeding using a dispenser, comprising: Steps for aspirating cell suspension using a dispenser; and The step of discharging the cell suspension to a fixed point in the culture container using the dispenser.

2. The method according to claim 1, wherein The suction step and the discharge step are performed once or twice or more, respectively.

3. The method according to claim 1, wherein The step of allowing the cells to adhere to the culture container by leaving the culture container at rest without shaking after the discharging step is further included.

4. The method according to claim 1, wherein The method further comprises the step of stirring the cell suspension before the aspirating step.

5. The method according to claim 1, wherein The method further comprises the step of stirring the cell suspension before the aspirating step, and then performing the aspirating step and the discharging step once or twice or more respectively.

6. The method according to claim 4 or 5, wherein: The aspirating step and the discharging step are completed after the stirring step and before the cells in the cell suspension settle.

7. The method according to claim 1, wherein The fixed point in the culture container is located at the center of the bottom surface of the culture container.

8. The method according to claim 1, wherein The culture container is a flat-bottomed culture container.

9. The method according to claim 1, wherein: The cells are stem cells.

10. The method according to claim 1, wherein The dispensing machine is an automated dispensing machine.

11. A method, which is a cell culture method in a culture container, comprising: The step of inoculating cells into a culture container using the method according to claim 1; as well as The step of culturing the cells in the culture container.

12. The method according to claim 11, wherein The method further comprises the step of inducing differentiation of the cells.

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