JP2023135601A - Cell collection method and cell collection device - Google Patents

Abstract

To provide a method of detaching and collecting cells from a base material using ultrasound, which is capable of effectively converting a cell mass detached from a base material into singulated cells with a high survival rate and also provide a cell collection device.SOLUTION: Provided is a method for collecting cells cultured on a base material. The method comprises the steps of: adding a cell detachment solution containing a metal ion chelating agent to a base material to which cells are attached and bringing the cells into contact with the cell detachment solution (Step 1); peeling off the cells from the base material by applying ultrasonic vibration to the base material and the cells (Step 2); and obtaining the cells that have been singulated with respect to the cells detached in the step 2 (Step 3). Also provided is a cell collection device capable of carrying out the cell collection method.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell collection method and a cell collection device.

In recent years, there has been a major paradigm shift in drug development, from conventional small molecule drugs and antibody drugs to cell therapy, which utilizes cells themselves as drugs, and regenerative medicine, which aims to regenerate tissues and organs. This cell medicine/regenerative medicine requires a large amount of cells, and in particular, it is required to efficiently and stably supply adhesive cells, which make up the majority of living tissues.

In culturing adherent cells, the desired cells are obtained through the following steps: culturing the cells on a culture substrate such as a polystyrene dish, peeling the cells from the substrate, collecting the cells, and washing them. . When it is desired to further increase the number of cells, a so-called passage operation is performed in which some or all of the obtained cells are transferred to a new substrate and cultured again. Among this series of steps, the peeling step of peeling cells from the substrate is a major barrier to efficient and stable supply of cells.

Generally, a method using proteolytic enzymes such as trypsin is widely used to detach cells from a substrate. In this method, proteins involved in the bond between cells and the substrate are degraded by the action of proteolytic enzymes, and then the cells are completely detached by operations such as tapping or pipetting. However, in this method, many other proteins on the cell surface are simultaneously degraded by proteolytic enzymes, leading to concerns that the quality of the cells may deteriorate.

On the other hand, a method is known in which cells are mechanically detached from a culture container without using proteolytic enzymes.

Non-Patent Document 1 proposes a cell detachment method using ultrasound. Non-Patent Document 1 discloses a method of peeling cells from a base material using a cell peeling device equipped with an ultrasonic transducer made of an annular piezoelectric element. According to the method described in Non-Patent Document 1, cells can be detached from a base material without using a proteolytic enzyme, so high quality cells can be provided.

Patent Document 1 proposes a mechanical cell detachment method that avoids the use of trypsin.
Furthermore, Patent Document 1 discloses that in order to convert cell aggregates peeled from a base material into single cells, a cell suspension containing cell aggregates is passed through a thin tube (inner diameter of 0.2 mm or more and 1.0 mm or less). The method is disclosed.

Patent No. 4775218

Yuta Kurashina. et al., Communications Biology, 2, 393 (2019)

In the cell detachment method described in Non-Patent Document 1, the cells detached from the base material may be in a state where the cells are associated with each other, forming a cell cluster. In the state of cell clusters, there are problems in that the subsequent proliferation efficiency decreases and cell counting becomes difficult.

In the method described in Patent Document 1, the tubules may become clogged if the cell clusters separated from the base material are large or if aggregates of cell clusters are present. In addition, there were cases in which the cells were insufficiently unified, and the survival rate of the unified cells was low.

Therefore, there has been a need for a more effective cell unification method for cells that have been mechanically detached from a substrate without using proteolytic enzymes, and for a simple device that can recover the unified cells.

An object of the present invention is to provide a cell collection method and a cell collection device that solve the problems in the background art described above.

In other words, in a method of detaching cells from a substrate using ultrasound and recovering single cells, it is possible to effectively convert cell clusters detached from the substrate into single cells with a high survival rate. An object of the present invention is to provide a cell collection method and a cell collection device that enable the cell collection.

A method for recovering cells according to one aspect of the present invention is a method for recovering cells cultured on a substrate, wherein a cell detachment solution containing a metal ion chelating agent is applied to the substrate to which the cells are attached. A step 1 in which the cells and the cell detachment solution are brought into contact with each other by adding a solution, and a step 2 in which the cells are detached from the base material by applying ultrasonic vibration to the base material and the cells. The present invention is characterized by comprising a step 3 of obtaining unified cells from the cells detached in step 2.

Further, a cell recovery device according to another aspect of the present invention is a cell recovery device for recovering cells cultured on a base material, wherein a metal ion chelating agent is added to the base material to which the cells are attached. an ultrasonic irradiation unit for applying ultrasonic vibrations to the base material and the cells; The present invention is characterized by comprising a singled cell acquisition unit that acquires the cells that have been unified with respect to the cells that have been peeled off from the material.

According to the present invention, in a method of detaching cells from a base material using ultrasound and recovering the unified cells, cell clusters detached from the base material are effectively unified with a high survival rate. It is possible to provide a cell recovery method and a cell recovery device that allow cell recovery.

1 is a flowchart illustrating an example of the flow of a cell collection method according to an embodiment of the present invention. FIG. 1 is a conceptual diagram of a cell collection device according to an embodiment of the present invention.

The present inventors have developed a method of detaching and recovering cells from a substrate using ultrasound without using proteolytic enzymes. We have carefully considered ways to obtain this. As a result, they found that the following method allows cells to be dissociated from cell aggregates while maintaining a high survival rate, and to effectively obtain single cells.

That is, this method uses a cell detachment solution containing a metal ion chelating agent as a cell detachment solution used when detaching cells from a substrate, and then detaches cells from the substrate using ultrasound. This is a method for obtaining single cells from a cell mass detached from a base material.

Here, as a method for obtaining the unified cells, there is a method of passing a cell suspension containing cell aggregates peeled from the base material through a mesh filter. Until now, mesh filters have been used to remove large debris and cell clumps that remain undissociated during enzymatic treatment of cells and tissues, and to isolate single cells. In an embodiment of the present invention, shear force is applied by passing a cell suspension containing cells detached from a base material through a mesh filter in a cell detachment solution containing a metal ion chelating agent. A mesh filter is used to provide the cell mass with the following properties: By using a mesh filter as described above in the embodiments of the present invention, it is possible to provide a novel cell collection method that can obtain unified cells without reducing cell survival rate.

Next, the present inventors investigated means for further reducing damage to cells caused by passing through a mesh filter. In the conventional method using proteolytic enzymes, cells can be easily unified, so after detaching the cells from the substrate using a physical method, the cells are removed from the shear force in the process of obtaining the unified cells. No effective means of protection has been found so far. As a result of extensive research, the present inventors found that by adding serum to a cell detachment solution (cell suspension) containing cells detached using ultrasound, the cells can be made resistant to shear force in subsequent steps. I found out that it is possible. As a result, they succeeded in converting cell clusters into single cells with a higher survival rate.
Embodiments of the present invention will be described in further detail below.

(effect)
The cell collection method according to the embodiment of the present invention includes a mechanical detachment method using ultrasonic vibration. What is important in the cell collection method according to the embodiment of the present invention is to have a process that unifies cells while peeling off cells adhered to a base material without damaging them.

Since the cell detachment solution contains a metal ion chelating agent, it can weaken the adhesion between adherent cells. As a result, when cells are detached by ultrasonic vibration, it becomes possible to detach the cells with small vibrations, and damage to the cells can be suppressed. Furthermore, this metal ion chelating agent can also contribute to cell unification in the subsequent step. That is, by weakening the adhesion between cells, it is possible to reduce the shear force on the cells that occurs when a cell mass is unified into cells.

As described below, it is particularly preferred to transform the cell mass into single cells after adding serum to the cell suspension. This is thought to be because proteins contained in serum have the function of protecting cells. As a result of the combination of the chemical separation effect of the metal ion chelating agent, the physical separation effect that imparts shear force to the cell aggregates, and the biological protection effect of the serum, cell aggregates obtained by mechanical detachment are particularly effective. It is speculated that the cells could be made into single cells.

A method for collecting cells according to an embodiment of the present invention is a method for collecting cells cultured on a substrate, and is characterized by having the following steps 1 to 3.
Step 1 is a step of adding a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached, and bringing the cells into contact with the cell detachment solution.
Step 2 is a step of exfoliating the cells from the base material by applying ultrasonic vibration to the base material and the cells.
Step 3 is a step of obtaining a single cell from the cells detached in Step 2.

(Cell culture conditions)
Cell culture conditions can be appropriately selected depending on the cells to be cultured. Generally, an appropriate medium is added to a dish as a substrate, cells are seeded therein at a density of about 1.0×10 ¹ to 5.0×10 ⁴ cells/cm ² , and the cells are incubated at 37°C and CO2. Culture in an environment with _{a 2} concentration of 5%. At this time, it is preferable to culture until the cell occupation area ratio in the substrate is approximately 70 to 80%, which is a so-called subconfluent state.

Below, steps 1 to 3 will be explained in detail.
(Step 1)
Step 1 in the cell collection method according to the embodiment of the present invention is a step of adding a cell detachment solution containing a metal ion chelating agent to the substrate to which the cells are attached and bringing the cells into contact with the cell detachment solution. be.

By bringing the cells into contact with the cell detachment solution, the bonds between cells and the adhesive force between the cells and the substrate are reduced. Through this step 1, cells can be exfoliated from the base material with high efficiency by the ultrasonic vibrations applied in the next step 2. In other words, if step 1 is not performed, cells cannot be detached with high efficiency.

The contact time between the cells and the cell detachment solution in step 1 is not particularly limited, but from the viewpoint of effectively detaching the cells and increasing the survival rate of the cells, it is, for example, 1 second to 24 hours, preferably 10 hours. The time period is from seconds to 12 hours, more preferably from 30 seconds to 6 hours.

The temperature of the cell detachment solution used in step 1 is not particularly limited, but from the viewpoint of maintaining cell viability, a temperature of 10°C or higher and 40°C is preferable, and particularly a temperature of 30°C or higher and 40°C is optimal for cell growth. This temperature is preferable.

Before and after step 1, cells may be washed with a buffer solution. For example, when you want to detach cultured cells from a substrate, you can first remove the growth medium and then wash the cells with a buffer solution while they are still attached to the substrate before adding cell detachment solution. , the effectiveness of the cell detachment solution may be increased. This is because growth media generally contain factors such as serum that inhibit cell detachment.

(cell)
The cells used in the embodiments of the present invention are not particularly limited as long as they can be cultured adhering to culture equipment in vitro. Examples of the cells include Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), and human fetal Various cultured cell lines such as kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, and human cervical cancer-derived HeLa cells; epithelial cells and endothelial cells that constitute various tissues and organs in the living body , skeletal muscle cells, smooth muscle cells, cardiomyocytes, which exhibit contractility, neuron cells that constitute the nervous system, glial cells, fibroblasts, hepatic parenchymal cells, hepatic non-parenchymal cells, and fat cells that are involved in the metabolism of the living body; Cells with differentiation potential include induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic cancer (EC) cells, mesenchymal stem cells, hepatic stem cells, and pancreatic stem cells. Examples include various stem cells such as stem cells, skin stem cells, muscle stem cells, and germline stem cells, and progenitor cells of various tissues; and cells induced to differentiate from cells with differentiation potential. Among these, the cell recovery method according to the embodiment of the present invention is suitable for cells with strong intercellular bonds, cells with high adhesion to a substrate, and cells with high trypsin sensitivity. It is particularly suitable for cells that tend to form cell clusters, such as mesenchymal stem cells.

(Base material)
The substrate in an embodiment of the present invention refers to a culture container used for cell culture. The culture container is not particularly limited as long as it is a cell-adhesive culture container, and examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, multi-well plates, multi-plates, etc. Examples include petri dishes, culture bags, and bottles.

The material of the base material in the embodiment of the present invention may be any material as long as it is chemically stable and capable of culturing desired cells. Examples of the base material include polyethylene, polypropylene, polycarbonate, polystyrene, polyvinyl chloride, nylon, polyurethane, polyurea, polylactic acid, polyglycolic acid, polyvinyl alcohol, polyvinyl acetate, poly(meth)acrylic acid, poly( Examples include meth)acrylic acid derivatives, polyacrylonitrile, poly(meth)acrylamide, poly(meth)acrylamide derivatives, polysulfone, cellulose, cellulose derivatives, polysilicone, polymethylpentene, glass, and metals. Among these, polystyrene is preferred as the material for the base material. Note that in this specification, the expression "(meth)acrylic" means "acrylic and methacrylic." For example, "poly(meth)acrylic acid" means "polyacrylic acid and polymethacrylic acid."

(Cell detachment solution containing metal ion chelating agent)
The cell detachment solution used in the embodiment of the present invention contains a metal ion chelating agent (hereinafter sometimes referred to as a chelating agent). By using a cell detachment solution containing a chelating agent, cells can be effectively detached from the substrate by ultrasonic vibration in step 2, and the treatment for obtaining unified cells in step 3 described below is also effective. can be done. If the cell detachment solution does not contain a chelating agent, the cell detachment rate will be low and the process to obtain unified cells will not be sufficient.

(chelating agent)
The chelating agent in the embodiment of the present invention is not particularly limited, but examples include ethylenediaminetetraacetic acid, ethylenediamine, ethylenediaminetetramethylenephosphonic acid, glycol etherdiaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, iminodiacetic acid, dihydroxyethyl Glycine, dicarboxymethylglutamic acid, ethylenediaminedisuccinic acid, etidronic acid, citric acid, gluconic acid, phosphonobutanetriacetic acid, and the like can be mentioned. Among these, as the chelating agent, a chelating agent that forms a chelate with a divalent cation is preferable, a chelating agent that forms a chelate with Ca ²⁺ and Mg ²⁺ is particularly preferable, and ethylenediaminetetraacetic acid is most preferable. When ethylenediaminetetraacetic acid is used as a chelating agent, the pH of the cell detachment solution is preferably 7.0 or more and 8.0 or less. This is because the pH is higher in the neutral range that can maintain a high cell survival rate, which increases the chelating ability of ethylenediaminetetraacetic acid and makes it possible to increase the peeling rate. be. Note that the chelating agent may be used alone or in combination of two or more types.

The content of the chelating agent in the cell detachment solution is preferably 0.01 mM or more and 5.0 mM or less. When the content of the chelating agent in the cell detachment solution is within this range, a chelating effect can be reliably obtained, and a decrease in cell activity due to the presence of an excessive chelating agent can be suppressed.

(Cell detachment solution)
The cell detachment solution used in the embodiment of the present invention is a solution containing a metal ion chelating agent, and is a solution used to detach cells from a substrate. The solution containing the metal ion chelating agent is not limited as long as it is an aqueous solution that does not impair cell activity, and may contain various salts, factors, and media. Furthermore, the cell detachment solution may contain serum and antibiotics. For example, the cell detachment solution may contain, as the above-mentioned factors, proteins such as albumin, insulin, and transferrin, selenium, antibiotics, sugars such as glucose, lipids, synthetic polymers, and the like, although there are no particular limitations thereon.

The pH of the cell detachment solution is preferably in the neutral range. This is because the neutral region is suitable for keeping cells stable, and the survival rate of cells can be kept stably high. The pH of the cell detachment solution can be adjusted as appropriate using hydrochloric acid, sodium hydroxide, or the like. Moreover, various buffer solutions are suitably used to keep the pH of the cell detachment solution stable.

The viscosity of the cell detachment solution is preferably 1.80 mPa·s or less. This is because the flow of the cell detachment solution generated by ultrasonic vibration is not hindered, and the detachment rate can be kept high. The viscosity of the cell detachment solution can be adjusted as appropriate by adding polymers or sugars.

The amount of protease in the cell detachment solution is preferably 0.0005% by mass or less based on the total mass of the cell detachment solution. Moreover, it is more preferable that the cell detachment solution does not substantially contain proteolytic enzymes. This is because, as mentioned in the background art, proteolytic enzymes decompose part of the cells, so if the cell detachment solution contains proteolytic enzymes, the detachment rate can be increased, but on the other hand, it may reduce the quality of the cells. This is because there is.

Here, the proteolytic enzyme is, for example, one that decomposes a part of the cell and makes it easier to separate the cell from the base material. Examples of proteases include trypsin, accutase, collagenase, natural protease, chymotrypsin, elastase, papain, pronase, and recombinant products thereof.

(buffer)
In an embodiment of the present invention, a buffer solution can be used in the cell detachment solution without any restrictions as long as it can maintain a neutral range. For example, buffers include Tris buffers such as Tris-HCl buffers, phosphate buffers, HEPES buffers, citric acid-phosphate buffers, glycylglycine-sodium hydroxide buffers, and Britton-Robinson buffers. , and GTA buffer. Among these, a phosphate buffer solution close to the in-vivo environment is preferred, and phosphate buffered saline (PBS) adjusted to be isotonic with intracellular fluid is more preferably used as the phosphate buffer solution.

(Culture medium)
There is no particular restriction on the type of medium that the cell detachment solution may contain; for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM. /F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM medium (alpha Modified Eagle's Minimum Essent) ial Medium; αMEM), MEM medium (Minimum Essential Medium ), RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William medium E, IPL41 medium, Fischer's medium, StemSpan H3000 (manufactured by Stem Cell Technology), StemS panSFEM (manufactured by Stem Cell Technology) ), Stemline II (manufactured by Sigma-Aldrich), Endothelial Cell Growth Medium 2 Kit (manufactured by Promocell), Mesenchymal Stem Cell Growth Medium 2 (manufactured by Promocell), MSCGM Bull et Kit (manufactured by Lonza), mTeSR1 or 2 medium (Stem Cell Technology) ), Repro FF or Repro FF2 (manufactured by Repro Cell), NutriStem medium (manufactured by Biological Industries), and MF-Medium mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.).
Among these, it is preferable to use a medium suitable for culturing each cell as the cell detachment solution.

(serum)
Examples of serum that the cell detachment solution may contain include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, and rabbit serum. serum, and human serum. Among them, FBS is easily used because of its easy availability. Further, the cell detachment solution may contain a serum-free medium containing purified blood-derived components or animal tissue-derived components (growth factors, etc.) without containing any untreated or unpurified serum.

(Antibiotics)
Examples of antibiotics that the cell detachment solution may contain include penicillin, streptomycin, ampicillin, carbenicillin, tetracycline, bleomycin, actinomycin, kanamycin, actinomycin D, amphotericin B, and the like.

(Step 2)
Step 2 is a step of peeling the cells from the base material by applying ultrasonic vibration to the cells. This step 2 makes it possible to effectively peel off cells that have adhered to the base material. In other words, unless step 2 is performed, cells cannot be detached with a high detachment rate.

Step 2 may include a step of applying an external stimulus such as convection to the cell detachment solution in addition to the step of applying ultrasonic vibration. Examples of methods for generating convection in the cell detachment solution include pipetting, a method using a pump, and a stirring blade.

There is no particular limitation on the time for applying ultrasonic vibration in step 2, but from the viewpoint of effectively detaching cells and increasing cell survival rate, for example, from 1 second to 1 hour, preferably from 5 seconds to 30 minutes. minutes, more preferably 10 seconds to 15 minutes.

The environmental temperature in step 2 is not particularly limited, but from the viewpoint of maintaining cell viability, it is preferably 20°C or more and 40°C or less, and more preferably 30.0°C or more and 37.5°C or less.
By setting the environmental temperature in step 2 to 20 to 40°C, the temperature during cell detachment will be close to the temperature during culture, reducing the effect of temperature changes on cells and maintaining a high cell survival rate. It's for a reason.

(Vibration caused by ultrasonic waves)
In the embodiment of the present invention, ultrasonic vibrations are vibrations with a frequency of 15 kHz or more. The vibration means can be used without particular limitation as long as it is capable of applying ultrasonic vibration to the substrate and cells. As an example, by bringing an ultrasonic vibrator containing a piezoelectric element such as lead zirconate titanate (PZT) into contact with a substrate on which cells are cultured, and inputting an arbitrary frequency, voltage, etc. to the ultrasonic vibrating member, Ultrasonic vibrations can be applied to the substrate and cells.
In this case, as the ultrasonic vibrator, a Langevin type vibrator, a flat plate vibrator in which a piezoelectric element is attached to a glass plate, a metal plate, etc. with an adhesive, etc. are preferably used. Note that a liquid such as water or a gel-like substance such as glycerol may be interposed between the ultrasonic transducer and the base material. By forming a layer of these liquid or gel-like substances, it is possible to prevent an air layer from forming between the ultrasonic transducer and the base material.

(Step 3)
Step 3 is a step of obtaining unified cells from the cells detached from the base material.
The treatment for obtaining single cells (hereinafter also referred to as single cell treatment) is not particularly limited as long as it does not include dissociation of cells by enzyme treatment, but it is possible to A preferred method is to pass it through a filter. In addition, it is more preferable that the single cell treatment is carried out with a cell suspension containing serum. For example, a cell suspension containing serum (which also contains a metal ion chelating agent) is The method of passing through a filter is a particularly preferred method. In addition, after adjusting the concentration of the metal ion chelating agent in the cell suspension containing the cells detached in step 2 above to be 1.0 mM or more, the single cellization treatment can be performed by applying shear force to the cells. It is also good to be done. For example, a single cell treatment may be performed by adding a highly concentrated metal ion chelating agent to a cell suspension containing cell aggregates peeled from a base material.

Each process will be explained in detail below.
(Single cell processing)
Here, the term "unified cell" refers to a cell that exists as a single cell (also referred to as a single cell) without aggregation among a plurality of cells. A single cell treatment is a treatment in which a cell group consisting of a plurality of cells, such as a cell cluster or a cell aggregate, is dissociated into individual cells.

(Single cell processing in which cell suspension is passed through a mesh filter)
An example of the single cell treatment in step 3 is performed by passing the cell suspension through a mesh filter.

For example, in the single-cell treatment, cells are mildly detached from a substrate using ultrasonic vibration using a cell detachment solution containing a metal ion chelating agent, and then a cell suspension containing the cell cluster detached from the substrate is used. This is done by passing the suspension through a mesh filter. Thereby, the cell mass is dissociated into individual cells while maintaining a high survival rate, and a single cell can be prepared.

The mesh filter used here is not particularly limited as long as it can be sterilized, and examples thereof include nylon mesh and metal mesh made of metal such as stainless steel. The pore size of the mesh should be any size that can convert cell clusters into single cells, and it depends on the type of cells, but the filter size should be selected appropriately depending on the size of the cells and the connectivity between cells. be able to.

The shape of the mesh in the mesh filter, that is, the shape of the pores through which cells pass, is not particularly limited as long as a single cell can pass therethrough. Therefore, the definition of the mesh size of the mesh filter differs depending on the shape of the mesh. For example, if the mesh shape is a square, the mesh size can be defined by the length of one side or the diagonal. When the shape of the mesh is rectangular, the mesh size can be defined by the long sides, short sides, diagonals, or the average length of these. When the shape of the mesh is an ellipse, the mesh size can be defined by the major axis, the minor axis, or the average length thereof. When the mesh shape is circular, the mesh size can be defined by the pore size (diameter). Mesh size can be measured by microscopic observation.
For example, the pore diameter of the nylon mesh is 10 μm or more and 100 μm or less, preferably 20 μm or more and 70 μm or less, particularly preferably 20 μm or more and 40 μm or less. In particular, by using a mesh filter with a pore size of 20 μm or more and 40 μm or less, it is possible to apply an appropriate shear force to the cell mass, and as shown in the examples below, cell unification and high survival rate can be achieved. Can be compatible.

Examples of methods for passing the cell suspension through a mesh filter include collecting the cell suspension from a culture dish and passing it through a mesh filter using a dispenser. By passing a cell suspension containing a metal ion chelating agent or containing a metal ion chelating agent and serum through a mesh filter, cell clusters are dissociated as they pass through the mesh filter, and cell unification is achieved. Ru. The cells in the obtained cell suspension are sufficiently unified and can be in a suitable state for the subsequent cell measurement process. Furthermore, the cell suspension containing the obtained single cells can be seeded again on a suitable culture substrate, thereby making it possible to effectively maintain and proliferate the obtained cells.

The number of times the cell suspension is passed through the mesh filter is not particularly limited as long as it is at least once, but it can be repeated as long as no damage to the cells occurs. It is also possible to combine mesh filter sizes. For example, it is also effective to first pass the cell suspension through a 40 μm mesh filter and then through a 20 μm mesh filter.

(Method of passing a cell suspension containing serum through a mesh filter)
An example of the single cell treatment in step 3 is performed by passing the serum-containing cell suspension through a mesh filter.

Using a cell detachment solution containing a metal ion chelating agent, cells are gently detached from the substrate using ultrasonic vibration, and then serum is added to the cell detachment solution. By passing the thus obtained cell suspension containing cell aggregates and serum through a mesh filter, the cell aggregates are dissociated while maintaining a high survival rate, and single cells can be prepared. The final concentration of serum in the cell suspension can be 0.1% or more and 90% or less, and is preferably 1% or more and 20% or less from the viewpoint of solution handling and serum cost. For example, a general cell culture medium often contains 10% serum, but when adding serum to the above cell detachment solution, add a cell culture medium containing an equal amount of serum to the cell detachment solution. This results in a final concentration of serum in the cell suspension of 5%. Serum at an appropriate concentration can contribute to improving cell survival rate.

The serum used here is not particularly limited as long as it contains serum-derived proteins that can be used in cell culture, and examples include serum itself and albumin, which is its main component. Examples of serum include FBS, horse serum, goat serum, and human serum.
FBS is easily used because it is easily available. As shown in the Examples below, the use of serum is effective for cell unification and high survival rate.

(Single cellization treatment performed using a metal ion chelating agent at a concentration of 1.0 mM or more)
An example of the single cell treatment in Step 3 is to adjust the concentration of the metal ion chelating agent in the cell suspension containing the cells detached in Step 2 above to 1.0 mM or more, and then share it with the cells. It is done by applying the force.

Using a cell detachment solution containing a metal ion chelating agent, cells are gently detached from the substrate using ultrasonic vibration, and then 1.0 mM or more is added to the cell suspension containing the cells detached from the substrate. Add metal ion chelating agent to the desired concentration.

The metal ion chelating agent added to the cell suspension after step 2 is not limited as long as it has the effect of dissociating bonds between cells, and examples thereof include ethylenediaminetetraacetic acid. When ethylenediaminetetraacetic acid is used as a chelating agent, the pH of the cell suspension is preferably 7.0 or more and 8.0 or less. This is because the pH is higher within the neutral range that can maintain a high survival rate, which increases the chelating ability of ethylenediaminetetraacetic acid and further promotes cell dissociation. .

The content of the chelating agent in the cell suspension is preferably 1.0 mM or more and 5.0 mM or less. By being within this range, a chelating effect can be reliably obtained, and a decrease in activity due to the presence of an excessive chelating agent can be suppressed.

In addition, this single cellization treatment involves adjusting the concentration of a metal ion chelating agent in the cell detachment solution to 1.0 mM or more, as well as applying shear force to the cells by creating convection in the cell detachment solution. including.

Examples of methods for causing convection in the cell detachment solution include pipetting, using a pump, and using a stirring blade. Shear force on the cells is applied, for example, by passing the cell suspension through a tubule. For example, a pipette tip can be used as a thin tube for passing the cell suspension. By applying shear force to the cells, it is possible to promote dissociation of cell clusters into single cells, and the influence of the metal ion chelating agent on the cells can be reduced. In order to further promote the singleization of cells from cell clusters, after the single cell treatment using a metal ion chelating agent at a concentration of 1.0 mM or more, the cell suspension was further passed through a filter mesh. Also good. The inner diameter of the tubule or the inner diameter of the pipette tip (the smallest part) should be 0.1 mm or more and 5 mm or less from the viewpoint of applying an appropriate shear force to the cells, maintaining survival rate, and water permeability of the cell suspension. It is preferably 0.15 mm or more and 2 mm or less.

(Flow of the cell collection method according to the embodiment of the present invention)
FIG. 1 shows an example of the flow of a cell collection method according to an embodiment of the present invention. For example, step 1 includes a step S10 of adding a cell detachment solution to the culture container, and a step S20 of incubating and maintaining the contact between the cells and the cell detachment solution for a predetermined period of time. Further, for example, the above step 2 includes a step S30 of applying ultrasonic vibration to detach the cells from the substrate, and further includes a step S40 of collecting a cell suspension containing the cells detached from the substrate. It's okay to stay. Further, for example, the step 3 includes step S50 of performing a single cell treatment. The flow of the cell collection method according to the embodiment of the present invention may further include a step of observing the state of the cells. Here, "incubating" refers to performing a step including at least one of adjusting temperature, adjusting humidity, and adjusting air composition. The step of adjusting the temperature is, for example, the step of controlling the heater so that the temperature around the cell culture container is around 37°C. The step of adjusting the humidity is, for example, a step of adjusting the humidity to 90% or more by installing a humidifying bat so that the culture medium does not dry out. The step of adjusting the air composition is, for example, a step of supplying CO ₂ so that the partial pressure of CO ₂ is around 5% in order to prevent oxidation of the medium.

(Configuration of cell collection device)
A cell recovery device according to an embodiment of the present invention is a cell recovery device that recovers cells cultured on a substrate, and includes a culture medium exchange section, an ultrasonic irradiation section, and a single cell acquisition section. have The exchange unit is configured to add a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached, and to bring the cells into contact with the cell detachment solution. Further, the ultrasonic irradiation unit is configured to apply ultrasonic vibration to the base material and the cells. Further, the unified cell acquisition unit is configured to acquire the unified cells from the cells peeled from the base material.

The configuration of the cell collection device 2 according to the embodiment of the present invention will be explained using the conceptual diagram shown in FIG. 2.
The culture substrate movement control unit 40 is a unit that holds and moves the culture substrate 44.
The culture substrate movement control unit 40 may be equipped with a heat insulator that controls the temperature and humidity of the environment in which cells are detached from the substrate. Examples include a unit and a humidifying unit. For example, an XYZ motorized stage is used to move the culture substrate 44. The culture substrate movement control unit 40 horizontally moves the culture substrate 44 between positions P4, P5, and P6. At position P4, an image of the cells cultured within the culture substrate 44 can be acquired. At position P5, the inside of the culture substrate 44 is irradiated with ultrasonic waves by the ultrasonic wave irradiator 1S. Further, at position P6, the solution in the culture substrate 44 is collected by the pipetting section 45, or various solutions are added to the culture substrate 44.

This will be further explained using an example of the process of cell detachment using ultrasound.
Cells are cultured on the culture substrate 44, and when it is time to detach the cells, the culture substrate 44 is placed at position P6. As appropriate, the culture substrate 44 can be placed at position P4 using the culture substrate movement control section 40, and the state of the cells can be observed using the cell observation section 43. Cells are attached to the culture substrate 44 placed at position P6.

First, it is necessary to remove the medium and replace it with cell detachment solution. Therefore, at position P6, the pipetting section 45 collects the culture medium in the culture substrate 44. The collected culture medium is discarded into the waste liquid collection section 47 at position P9. Various media are stored in a washing medium 50, a culture medium 51, and a cell detachment solution 52, and are transported to the pipetting section 45 by a liquid transport control section 53. The pipetting section 45 can access the culture substrate 44 by moving up and down. As appropriate, the cell collection device 2 may include a system for opening and closing the lid of the culture substrate 44.

After removing the medium, cells adhering to the culture substrate 44 can be washed as appropriate.
The liquid transport control section 53 transports the washing medium 50 to the pipetting section 45, and the pipetting section 45 adds the washing medium 50 into the culture substrate 44 at position P6. After removing the washing medium 50 in the same manner as the above-described medium removal method, the liquid transport control section 53 transports the cell detachment liquid 52 to the pipetting section 45 . Subsequently, at position P6, the cell detachment solution 52 is added into the culture substrate 44 by the pipetting unit 45.

Next, the culture substrate movement control unit 40 moves the culture substrate 44 to position P5. Here, the culture substrate 44 is set in the ultrasonic irradiation section 1S. A conventionally known configuration can be used as the ultrasonic irradiation section 1S. The configuration including the ultrasonic irradiation section 1S includes an ultrasonic peeling unit 54 having the ultrasonic irradiation section 1S, a function generator 42 and an amplifier 41 for inputting arbitrary frequencies, voltages, etc. to the ultrasonic peeling unit 54. An example is a combination of the following.

The culture substrate 44 is irradiated with ultrasonic waves from the ultrasonic irradiation unit 1S, and cells adhering to the culture substrate 44 are peeled off. Since the cells detached from the culture substrate 44 contain cell clusters, they are next subjected to a single cell treatment.

First, the culture substrate movement control unit 40 moves the culture substrate 44 to position P6. At position P6, the pipetting unit 45 collects a cell suspension containing cells detached from the culture substrate 44 within the culture substrate 44. The collected cell suspension is passed through a mesh filter F2 at position P10, and then seeded onto a new cell culture substrate installed at the exfoliated cell collection section 48, for example, position P7. Alternatively, at position P10, the cell suspension is passed through mesh filter F2 and collected into tube 49, which is exfoliated cell collection section 48. The collected cells are unified and used for subsequent assays such as cell culture and cell counting.

The cell collection device 2 may include a cell observation unit 43 that acquires images of cells cultured within the culture substrate 44, allowing the state of cell detachment and the state of cell collection to be confirmed.

The cell collection device 2 may include a control section that controls the above cell detachment process. The control section is a means for controlling the operation of each section within the cell collection device 2. For example, the control unit may be configured by a computer having an arithmetic processing unit such as a CPU, a memory such as a RAM, and a storage unit such as a hard disk drive. The control section may include an operation section.
The operation unit may include a display unit, an input unit, and the like. The display unit can display image data output from the control unit and various information related to the operation of the cell collection device 2. For example, a liquid crystal display is used for the display section. The input unit receives various instruction inputs from the user. For example, a keyboard or a mouse is used as the input unit. Both the display section function and the input section function may be provided in a single unit such as a touch panel display.

Hereinafter, the present invention will be explained in more detail using Examples and Comparative Examples, but the Examples and Comparative Examples described below do not limit the present invention in any way.

<Preparation example of cell detachment solution 1>
Add 1.0% by mass of a hydrophilic polymer and 0.5 mmol/L of ethylenediaminetetraacetic acid to phosphate buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) that does not contain divalent cations. It was added to the desired concentration and completely dissolved. As the hydrophilic polymer, PEG2000 (manufactured by Kishida Chemical Co., Ltd.) having a peak molecular weight Mp of 2030 measured by GPC was used. Furthermore, the pH was adjusted to 7.4 using hydrochloric acid and sodium hydroxide, and this was used as cell detachment solution 1. The viscosity of cell detachment solution 1 was 1.22 mPa·s.

<Preparation example of cell detachment solution 2>
Add 1.0% by mass of a hydrophilic polymer and 0.1 mmol/L of ethylenediaminetetraacetic acid to phosphate buffered saline (PBS(-), manufactured by Thermo Fisher Scientific) that does not contain divalent cations. It was added to the desired concentration and completely dissolved. As the hydrophilic polymer, PEG4000 (manufactured by Kishida Chemical Co., Ltd.) having a peak molecular weight Mp of 3700 as measured by GPC was used. Furthermore, the pH was adjusted to 7.4 using hydrochloric acid and sodium hydroxide, and this was used as cell detachment solution 2. The viscosity of cell detachment solution 2 was 1.23 mPa·s.

[Example 1]
(Culture of cells on substrate)
hMSC (Human Mesenchymal Stem Cells) cells were seeded in a Φ60 polystyrene dish (manufactured by Corning) at a density of 4000 cells/cm ² and cultured at 37° C. in an environment with a CO ₂ concentration of 5%. The medium used was MSCGM Bullet Kit (manufactured by Lonza). After 96 hours of culture, the medium was replaced, and then culture was continued for 168 hours. Cells were observed using a phase contrast microscope to confirm cell adhesion and proliferation. The cell occupation area ratio of the dish was about 80%.

(Detachment of cells from base material)
The medium in the dish was removed, the cells were washed with PBS(-), and then immersed in the above cell detachment solution 1 for 10 minutes. Thereafter, the dish was set in an ultrasonic peeling device, and a sweep vibration (frequency 22-27 kHz, sweep period 1 s, voltage 100 V) was applied for 3 minutes at an environmental temperature of 37° C. to peel the cells from the substrate.

(Single cell processing)
Aspirate the cell suspension in which cell clumps are suspended in cell detachment solution 1 with a pipettor, and gently press the tip of the pipette against a mesh filter (mesh size 20 μm) that has been set in a 15 mL tube in advance. The suspension was passed through a mesh filter. Thereafter, the cell suspension that had passed through the filter was collected.

When the obtained cell suspension was placed in a hemocytometer and four fields of view (one field of view was 1 mm ² ) were observed under a microscope, no cell clusters were observed. As a result of calculating the cell viability using a hemocytometer to measure the number of cells and a live/dead determination method using trypan blue staining, the cell viability was 80%. Therefore, single cell processing using a mesh filter was possible.

(Comparative example 1)
As in Example 1, cells were detached from the dish by applying ultrasonic vibration. A cell suspension in which cell aggregates were suspended in cell detachment solution 1 was placed in a hemocytometer, and when four fields of view were observed under a microscope, many cell aggregates were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 1, the cell survival rate was 79%. When no single cellization treatment was performed, no single cells were obtained. Therefore, it was difficult to measure the number of cells.

(Example 2)
As in Example 1, cells were detached by applying ultrasonic vibration. Subsequently, before performing the single cell treatment, serum (a solution containing 10% serum, i.e., MSCGM Bullet Kit) was added to the cell suspension in which the cell aggregates were suspended in cell detachment solution 1. . The final concentration of serum in the cell suspension was now 5%. Next, the cell suspension was aspirated with a pipetter, and the cell suspension was passed through the mesh filter (mesh size 20 μm), which had been set in a 15 mL tube while lightly pressing the tip of the pipette. Thereafter, the cell suspension that had passed through the mesh filter was collected.

When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. As a result of calculating the cell viability using a hemocytometer to measure the number of cells and a live/dead determination method using trypan blue staining, the cell viability was 90%. Therefore, it was possible to unify cells using a mesh filter. In the single cell treatment, it was possible to improve cell survival rate by adding serum.

(Example 3)
In Example 2, cell detachment and single cell treatment were performed in the same manner as in Example 2, except that the mesh size of the mesh filter was 10 μm. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 83%. Therefore, it was possible to unify cells using a mesh filter.

(Example 4)
In Example 2, cell detachment and single cell treatment were performed in the same manner as in Example 2, except that the mesh size of the mesh filter was 30 μm. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 93%. Therefore, it was possible to unify cells using a mesh filter.

(Example 5)
In Example 2, cell detachment and single cell treatment were performed in the same manner as in Example 2, except that the mesh size of the mesh filter was 40 μm. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 94%. Therefore, it was possible to unify cells using a mesh filter.

(Example 6)
In Example 2, cell detachment and single cell treatment were performed in the same manner as in Example 2, except that the mesh size of the mesh filter was 50 μm. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, some single cells and cell clusters with a diameter of 50 μm or more were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 92%.

(Example 7)
In Example 2, cell detachment and single cell treatment were performed in the same manner as in Example 1, except that the mesh size of the mesh filter was 70 μm. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, some single cells and cell clusters with a diameter of 50 μm or more were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 90%.

(Comparative example 2)
As in Example 2, cells were detached from the dish by applying ultrasonic vibration. Thereafter, serum was added to the cell suspension in which the cell aggregates were suspended in cell detachment solution 1, and the cells were collected. Thereafter, the cell suspension was placed in a hemocytometer without passing it through a mesh filter, and when four fields of view were observed under a microscope, many cell clumps were observed. Therefore, cells could not be unified. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 2, the cell survival rate was 92%.

(Example 8)
The cell suspensions obtained after the single cell treatment in Examples 2 to 4 and 6 and after the detachment of cells from the dishes in Comparative Example 2 were seeded onto new substrates (Corning dishes). At this time, the number of cells to be seeded was set to 10,000. Thereafter, the cells were cultured for 5 days at 37° C. and a CO ₂ concentration of 5%. After culturing, the cells were detached from the substrate using trypsin, and the number of cultured cells was measured.
The results are shown in Table 1.

Note that the mesh size in Table 1 indicates the mesh size of the mesh filter used in the single cell processing in each Example. Furthermore, the number of cells after culture in Table 1 indicates the number of cells after 5 days of culture.

As is clear from Table 1, the cells obtained through the single cell treatment using the mesh filter in Examples 2 to 4 and 6 were not only unified, but also had no problems with proliferation during subsequent culture. I couldn't see it. Compared to Comparative Example 2 in which no single cell treatment using a mesh filter was performed, a higher number of cells was obtained in Example 2 using a filter mesh with a mesh size of 20 μm and in Example 4 using a filter mesh with a mesh size of 30 μm. was gotten.

(Example 9)
As in Example 1, cells were detached from the dish by applying ultrasonic vibration. Before carrying out the single-cell treatment, ethylenediaminetetraacetic acid for single-cell treatment was added to a cell suspension in which cell aggregates were suspended in cell detachment solution 1. The final concentration of ethylenediaminetetraacetic acid in the cell suspension was 5mM. After adding ethylenediaminetetraacetic acid to the cell suspension, it was left at 37°C for 15 minutes. Next, the cell suspension was stirred by pipetting.
When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 1, the cell survival rate was 92%. Therefore, it was possible to unify cells using ethylenediaminetetraacetic acid.

(Comparative example 3)
In Example 9, cell detachment and cell unification treatment (pipetting operation only) were carried out in the same manner as in Example 9, except that ethylenediaminetetraacetic acid for single cellization treatment was not added to the cell suspension. I did this. The detached cells were cell clusters, and no single cells were obtained.

(Comparative example 4)
Cells were detached in the same manner as in Example 1, except that PBS(-) was used instead of cell detachment solution 1. As a result, the cells hardly peeled off from the substrate.
Further, some of the detached cells were cell clusters, and no single cells were obtained.
From this, it can be seen that in the cell collection method according to the example of the present invention, the cell detachment solution used in step 1 needs to contain a metal ion chelating agent.

(Comparative example 5)
In Example 1, cells were detached in the same manner as in Example 1 except that no ultrasonic vibration was applied, but the cells were hardly detached from the base material. Further, some of the detached cells were cell clusters, and no single cells were obtained.
From this, it can be seen that the ultrasonic vibration used in step 2 is necessary in the cell collection method according to the example of the present invention.

(Example 10)
Mouse skeletal muscle myoblasts (hereinafter abbreviated as C2C12 cells) were seeded in a Φ35 polystyrene dish (manufactured by Corning) at a density of 10,000 cells/cm ² and cultured at 37° C. in an environment with a CO ² concentration of 5%. The medium used was DMEM (manufactured by Thermo Fisher Scientific) to which the following two materials were added.
・Fetal Bovine Serum (manufactured by Sigma-Aldrich), final concentration 10% by mass
・Penicillin-streptomycin (10000 U/ml, manufactured by Thermo Fisher Scientific), final concentration 1% by mass
Culture was carried out for 48 hours, and the state of the cells was observed using a phase contrast microscope to confirm cell adhesion and proliferation. The cell occupation area ratio of the dish was about 80%.
Next, cells were detached in the same manner as in Example 2, except that the cells in Example 2 were changed to C2C12 cells, the cell detachment solution was changed to Cell Dissection Solution 2, and the serum-containing solution was changed to DMEM containing 10% serum. and single cell treatment was performed. When the obtained cell suspension was placed in a hemocytometer and four fields of view were observed under a microscope, no cell clusters were observed. Furthermore, as a result of calculating the cell survival rate in the same manner as in Example 1, the cell survival rate was 85%. Therefore, it was possible to unify cells using a mesh filter. Furthermore, when mesh filters with mesh sizes of 40 μm and 70 μm were used, similar survival rates (86% and 87%, respectively) were obtained, and no cell clusters were observed.

The disclosure herein includes the following methods and configurations.
(Method 1)
A method for collecting cells cultured on a substrate, the method comprising:
Step 1 of adding a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached and bringing the cells into contact with the cell detachment solution;
Step 2 of peeling off the cells from the base material by applying ultrasonic vibration to the base material and the cells;
Step 3 of obtaining the cells that have been unified with respect to the cells detached in Step 2;
A method for collecting cells characterized by comprising:
(Method 2)
The method for recovering cells according to method 1, wherein step 3 includes passing the cell suspension containing the cells detached in step 2 through a mesh filter.
(Method 3)
The method for collecting cells according to method 2, wherein the mesh filter has a mesh size of 20 μm or more and 40 μm or less.
(Method 4)
The step 3 includes applying a shear force to the cells after adjusting the concentration of the metal ion chelating agent in the cell suspension containing the cells detached in the step 2 to be 1.0 mM or more. , Method 1 for collecting cells.
(Method 5)
4. A method for recovering cells according to method 4, wherein the shear force is provided by passing the cell suspension through a capillary.
(Method 6)
The method for collecting cells according to method 5, wherein the tubule is a pipette tip.
(Method 7)
7. The method for collecting cells according to any one of methods 1 to 6, which comprises adding serum to a cell suspension containing the cells detached in step 2, before step 3.
(Method 8)
The cell detachment solution contains a metal ion chelating agent of 0.01 mM or more and 5.0 mM or less,
A method for collecting cells according to any one of methods 1 to 7.
(Method 9)
9. The method for recovering cells according to any one of methods 1 to 8, wherein the cell detachment solution does not contain a proteolytic enzyme.
(Method 10)
The method for collecting cells according to any one of methods 1 to 9, wherein the cells are stem cells.
(Method 11)
The method for collecting cells according to any one of Methods 1 to 10, which is carried out under conditions where the environmental temperature is 30.0° C. or higher and 37.5° C. or lower.
(Configuration 1)
A cell collection device for collecting cells cultured on a substrate,
a medium exchange section that adds a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached, and brings the cells into contact with the cell detachment solution;
an ultrasonic irradiation unit for applying ultrasonic vibration to the base material and the cells;
a unified cell acquisition unit that acquires the cells that have been unified with respect to the cells that have been detached from the base material;
A cell collection device characterized by having the following.
(Configuration 2)
The cell collection device according to configuration 1, wherein the single cell acquisition unit includes a mesh filter.

2 Cell recovery device 1S Ultrasonic irradiation unit 40 Culture substrate movement control unit 41 Amplifier 42 Function generator 43 Cell observation unit 44 Cell culture substrate 45 Pipetting unit 46 Pipetter movement control unit 47 Waste liquid recovery unit 48 Detachable cell recovery unit (cell culture substrate)
49 Detachment cell collection section (tube)
50 Washing medium 51 Culture medium 52 Cell detachment liquid 53 Liquid transport control section 54 Ultrasonic detachment unit F2 Filter for cell unification

Claims (13)

A method for collecting cells cultured on a substrate, the method comprising:
Step 1 of adding a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached and bringing the cells into contact with the cell detachment solution;
Step 2 of peeling off the cells from the base material by applying ultrasonic vibration to the base material and the cells;
Step 3 of obtaining the cells that have been unified with respect to the cells detached in Step 2;
A method for collecting cells characterized by comprising: The method for collecting cells according to claim 1, wherein the step 3 includes passing the cell suspension containing the cells detached in the step 2 through a mesh filter. The method for collecting cells according to claim 2, wherein the mesh filter has a mesh size of 20 μm or more and 40 μm or less. The step 3 includes applying a shear force to the cells after adjusting the concentration of the metal ion chelating agent in the cell suspension containing the cells detached in the step 2 to be 1.0 mM or more. , The method for collecting cells according to claim 1. 5. The method of recovering cells according to claim 4, wherein the shear force is applied by passing the cell suspension through a capillary. The method for collecting cells according to claim 5, wherein the thin tube is a pipette tip. 2. The method for recovering cells according to claim 1, comprising adding serum to a cell suspension containing the cells detached in step 2, before the step 3. The cell detachment solution contains a metal ion chelating agent of 0.01 mM or more and 5.0 mM or less,
The method for collecting cells according to claim 1. The method for collecting cells according to claim 1, wherein the cell detachment solution does not contain a proteolytic enzyme. The method for collecting cells according to claim 1, wherein the cells are stem cells. The method for collecting cells according to claim 1, wherein the method is carried out at an environmental temperature of 30.0°C or higher and 37.5°C or lower. A cell collection device for collecting cells cultured on a substrate,
a medium exchange part that adds a cell detachment solution containing a metal ion chelating agent to the base material to which the cells are attached, and brings the cells into contact with the cell detachment solution;
an ultrasonic irradiation unit for applying ultrasonic vibration to the base material and the cells;
a unified cell acquisition unit that acquires the cells that have been unified with respect to the cells that have been detached from the base material;
A cell collection device characterized by having the following. The cell collection device according to claim 12, wherein the single cell acquisition unit includes a mesh filter.

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