JP2022105806A - Method for culturing - Google Patents

Abstract

To provide a culturing method using a temperature-responsive culture vessel, enabling a cooling system for the culture vessel to be made compact and automatic.SOLUTION: A method for an adherent cell includes the steps of (a) culturing an adherent cell in a temperature-responsive culture vessel, (b) recovering the medium from the culture vessel, (c) cooling the recovered medium, and (d) adding the cooled medium to the culture vessel.SELECTED DRAWING: None

Description

The present invention relates to a method for culturing adherent cells using a temperature-responsive culture equipment.

Since a large amount of cells are required for the production of cell medicines, a highly efficient subculture method is required. Many raw material cells of cell medicines require adhesion to a base material that serves as a scaffold for proliferation, but when recovering the grown adherent cells, it is necessary to detach the cells from the substrate.

As a method for exfoliating cells from a substrate, there are generally a method of treating with a proteolytic enzyme such as trypsin and a method of using a temperature-responsive culture device. When a temperature-responsive culture device is used, the cells are exfoliated by weakening the adhesive force on the surface of the device by performing a cooling treatment (for example, Patent Document 1 and Patent Document 2). Recovery of cells by such a cooling treatment has an advantage that cells with low invasiveness and high quality can be obtained as compared with treatment with a proteolytic enzyme.

However, the cooling treatment in the conventional method is by cooling the entire temperature-responsive culture equipment or exchanging the medium of the culture equipment with a cooled medium, and a large-volume cooling device is required. There was room for improvement in terms of points and points that were difficult to automate.

JP-A-2018-87316 Japanese Unexamined Patent Publication No. 2020-152874

The present invention has been made in view of such a situation, and an object thereof is a method for culturing adherent cells using a temperature-responsive culture equipment, and the cooling system of the culture equipment is miniaturized and automated. Is to provide a possible way.

As a result of diligent research, the present inventors have collected a medium from a temperature-responsive culture device in which adherent cells are cultured, cooled the medium, and then returned the culture medium to the culture device. We have found that it is suitable for miniaturization and automation of cells and enables highly efficient culture for cell passage, and have completed the present invention.

That is, the present invention includes the following aspects.

[1] A method for culturing adherent cells.
(A) A step of culturing adherent cells in a temperature-responsive culture device,
(B) A step of recovering the medium from the culture equipment,
(C) A step of cooling the recovered medium, and (d) a step of adding the cooled medium to the incubator.
How to include.

[2] The method according to [1], wherein the adherent cells are proliferated above the subconfluent in step (a).

[3] The method according to [1] or [2], wherein a liquid feed pump is used to recover the medium in the step (b).

[4] The method according to any one of [1] to [3], wherein a water-cooled cooler is used to cool the medium in the step (c).

[5] The method according to any one of [1] to [4], wherein a liquid feed pump is used for adding the cooled medium in the step (d).

[6] The method according to any one of [1] to [5], which is a closed system.

According to the culture method of the present invention, the temperature-responsive culture equipment can be cooled by cooling the minimum necessary medium recovered from the culture equipment, so that a small cooling device can be used. .. Further, since the collection and cooling of the medium can be performed by introducing a pump and a refrigerant, automation is easy. Therefore, the adherent cells for passage can be cultured with high efficiency.

It is a figure which shows the culture system used in an Example.

In the method for culturing adherent cells of the present invention, first, the adherent cells are cultured in a temperature-responsive culture device (step (a)).

In the present invention, the "adherent cell" means a cell that proliferates while adhering to a substrate. Examples of the adherent cells include Chinese hamster ovary-derived CHO cells, mouse binding tissue L929 cells, human fetal lung-derived normal diploid fibroblast cells (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), and human offspring. In addition to various cultured cell lines such as HeLa cells derived from cervical cancer, for example, epithelial cells and endothelial cells constituting each tissue or organ in the living body; contractile skeletal muscle cells, smooth muscle cells, myocardial cells; nervous system Neuronal cells, glia cells, fibroblasts; hepatic parenchymal cells, non-hepatic parenchymal cells, fat cells involved in biological metabolism; induced pluripotent stem (iPS) cells with differentiation potential, embryonic stem (ES) ) Cells, embryonic reproductive (EG) cells, embryonic cancer (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, reproductive stem cells and other stem cells and precursor cells of each tissue, they Examples thereof include cells induced to differentiate from stem cells and precursor cells. In addition to these, cells (living cells) contained in blood, lymph, cerebrospinal fluid, sputum, urine or stool, microorganisms, viruses, protozoans and the like existing in the body or environment can be exemplified.

In the present invention, the "temperature-responsive culture equipment" is a culture equipment coated with a polymer, and when cooled to a temperature lower than the lower limit critical dissolution temperature (LCST; Lower Critical Solution Temple) of the polymer. , Means a culture device in which the polymer dissolves. By dissolving the polymer on the surface, the cells adhering to the culture equipment can be exfoliated. Here, the "lower limit critical dissolution temperature" means that at a temperature lower than this temperature, the polymer dissolves in water and becomes a transparent solution, but at a temperature higher than this temperature, it becomes insoluble and becomes cloudy or precipitates. The temperature at which phase separation occurs.

Examples of the repeating unit of a polymer having the characteristics of LCST (hereinafter referred to as “temperature-responsive polymer”) and its water are as follows.

N-ethylacrylamide (LCST = 72 ° C), N-cyclopropylacrylamide (LCST = 46 ° C), N-isopropylacrylamide (LCST = 32 ° C), Nn-propylmethacrylamide (LCST = 22 ° C), N- Tetrahydrofurfurylacrylamide (LCST = 28 ° C), N-ethoxyethylacrylamide (LCST = 35 ° C), N, N-diethylacrylamide (LCST = 32 ° C), N-cyclopropylmethacrylamide (LCST = 59 ° C), N -Isopropylmethacrylamide (LCST = 44 ° C.), Nn-propylmethacrylamide (LCST = 28 ° C.), N-tetrahydrofurfurylmethacrylamide (LCST = 35 ° C.), N-methyl-N-ethylacrylamide (LCST =) 56 ° C.), N-methyl-N-isopropylacrylamide (LCST = 23 ° C.), N-methyl-Nn-propylacrylamide (LCST = 20 ° C.), or N, N-dimethylaminoethyl methacrylate (LCST = 47 ° C.) ).

In the temperature-responsive culture equipment, the temperature-responsive polymer may be covalently linked to the surface of the substrate, or the temperature-responsive polymer may be coated by an interaction other than the covalent bond. Examples of the former include UpCell (CellSeed), and examples of the latter include cell culture equipment coated with the block copolymer described in Japanese Patent No. 6638706 and Japanese Patent No. 5846584. The shape of the incubator is not particularly limited, and examples thereof include a petri dish, a flask, a plate, a bag, and particles.

The surface of the culture equipment may be further coated with an extracellular matrix. The type of extracellular matrix is not particularly limited, and for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glucosaminoglycan, fibronectin, laminin, vitronectin, gelatin, laminin, collagen IV, heparan sulfate proteoglycan, entactin / nidogen 1 A matrix gel containing 1, 2 or the like as a main component may be used, or only one type of these may be used, or two or more types may be combined. It may also be a segment of these extracellular matrices.

The adherent cells in this step are cultured at a temperature higher than that of the surface-treated polymer LCST in order to proliferate the cells while adhering to the culture equipment. In this step, it is preferable to proliferate the adherent cells above the subconfluent. As a result, the culture efficiency can be improved when the cells collected by the culture method of the present invention are transferred to a new culture equipment and subcultured in which the cells are cultured again. Here, the "subconfluent" refers to a state in which 60 to 90% of the adhesive surface of the culture vessel is occupied by cells, and there is still room for cells to grow. Therefore, "subconfluent or higher" in the present invention means a state in which adherent cells occupy 60% or more of the adherent surface of the culture vessel.

The medium of the adherent cells is not particularly limited as long as the cells are suitable for adhering to the culture equipment and proliferating. The medium generally consists of basal medium and serum, but may contain other elements such as antibiotics. The type of basal medium is not particularly limited, and for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, RPMI1640 and the like can be used. can. The type of serum is not particularly limited, and for example, bovine fetal serum, calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum are used, but are available. Generally, bovine fetal serum is often used because of its ease of use. The serum concentration in the medium is not particularly limited, and is, for example, 1 to 30 vol%. Further, the medium may be a serum-free medium containing no untreated or unpurified serum and containing purified blood-derived components or animal tissue-derived components (growth factors, etc.).

In the culture method of the present invention, the medium is then recovered from the culture equipment (step (b)).

The method for recovering the medium from the culture equipment is not particularly limited, but it is preferably mechanized using a liquid feed pump in order to carry out efficient subculture. The amount of liquid to be fed is not particularly limited, but is preferably 0.1 to 100.0 mL / min in order to efficiently cool the medium in the step (c) described later. The type of liquid feed pump is not particularly limited, but a tube pump is preferable because it is easy to adjust the liquid feed amount and a plastic tube can be applied. The material of the tube is not particularly limited, but it is preferably made of plastic because the medium contains reducing sugar. The inner diameter of the tube is not particularly limited, and is, for example, 0.1 to 30.0 mm.

In the culture method of the present invention, the recovered medium is then cooled (step (c)).

As a method for cooling the culture equipment, a method of cooling the culture equipment in a cold place has been conventionally used, but the volume of the cooler becomes large and it is difficult to perform efficient subculture by automation. be. In addition, a method of extracting the medium from the culture equipment and adding a new cooled medium is also known, but since it is necessary to prepare a cooled medium separately and the workability is low, this method is also an efficient passage. Culturing is difficult. On the other hand, the method of the present invention is characterized in that the medium is recovered from the temperature-responsive culture equipment in which the adherent cells are cultured, cooled, and then returned to the culture equipment, so that a large cooling system is not required. It is suitable for automation and enables highly efficient culture for cell passage.

The method for cooling the recovered medium is not particularly limited, but it is preferable to cool the collected medium with a water-cooled cooler having high thermal conductivity. A simple cooling system may be used to facilitate maintenance. For example, when a liquid feed pump (for example, a tube pump) is used for collecting the medium in step (b), the tube is immersed in a cooling tank. May be good. On the other hand, in order to further improve the cooling efficiency, a coiled tube may be used, for example, a Graham condenser or a Dimroth condenser. Since the temperature of the medium added to the culture equipment in the subsequent step (d) needs to be lower than the LCST of the polymer covering the surface of the temperature-responsive culture equipment, the cooling temperature is usually set. , The temperature is 2 ° C. or higher, preferably 5 ° C. or higher lower than the LCST. Since the main component of the medium is water, the lower limit of the cooling temperature is the temperature at which the medium does not freeze (typically 0 ° C.). The type of cooling water is not particularly limited, and for example, water, antifreeze water such as ethylene glycol can be used.

In the culture method of the present invention, a cooled medium is then added to the culture equipment (step (d)).

This step can also be said to be a step of returning the medium to the culture equipment from which the medium has been collected in a cooled state. There is no particular limitation on the method of adding the medium to the incubator. When a liquid feed pump (for example, a tube pump) is used, the above-mentioned recovery of the medium (step (b)) and cooling of the medium (step (c)) can be continuously performed. That is, the operation of immersing one end of the tube installed in the liquid feed pump in the culture equipment to collect the medium, cooling it, and then returning the cooled medium from the other end of the tube to the culture equipment. It can be circulated (see Figure 1). After stopping the circulation of this medium, the medium may remain in the tube, but when adding the entire amount of the recovered medium to the early culture equipment, for example, raise the end of the tube into the air layer. The medium in the tube can be discharged to the culture equipment. Alternatively, the end of the tube may be connected to a liquid phase such as fresh medium and the medium in the tube may be discharged to the culture medium. As a result, the temperature-responsive culture equipment is cooled to a temperature lower than the LCST of the polymer covering the surface thereof, and the cells are detached from the surface of the culture equipment over time. The time until peeling may vary depending on the type of the polymer and the coating amount, but is, for example, 5 to 60 minutes. For detachment of adherent cells from the culture equipment, in addition to the method of waiting for such natural detachment, for example, a method of giving a physical impact to the entire equipment (for example, tapping or shaking) or a method of giving a local impact to the cells. A method (eg, a cell scraper method or pipetting) can be used, which can reduce the time to detachment of adherent cells.

The detached cells may be collected manually using a pipette or the like, or mechanically using a pump or the like. When a pipette is used, cells can be efficiently collected by forming a cell suspension by operating the medium in and out of the pipette. When forming the cell suspension, it is preferable to operate gently in order to suppress foaming.

The cells thus collected can be subcultured by transferring to a new culture equipment and culturing. Therefore, the present invention comprises a step of recovering cells exfoliated from the temperature-responsive culture equipment (step (e)) and a step of transferring the recovered cells to a new temperature-responsive culture equipment and culturing them (step (f)). Further provided are methods for subculturing adherent cells, including.

The culture method of the present invention is preferably a closed system in order to prevent substances that should not be originally mixed (so-called contamination) from being mixed in the culture equipment. Since the culture method of the present invention is suitable for mechanical operation as compared with the culture method using the conventional temperature-responsive culture equipment, there is an advantage that it is easy to construct as a closed system.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples. Unless otherwise noted, commercially available reagents were used.

[Materials and methods]
(1) Composition of temperature-responsive block copolymer The composition of the temperature-responsive block copolymer is proton nuclear magnetic resonance spectroscopy (manufactured by JEOL Ltd., trade name: JNM-ECZ400S / LI). ¹ H-NMR) Obtained from spectral analysis.

(2) Molecular weight and molecular weight distribution of temperature-responsive block copolymers Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) are measured by gel permeation chromatography (GPC). did. As the GPC apparatus, HLC-8320GPC (manufactured by Tosoh Corporation) was used, and as the column, two TSKgel Super AWM-H (manufactured by Tosoh Corporation) were used. The column temperature was set to 40 ° C. and the eluent was measured using 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg / mL and measured. For the calibration curve of the molecular weight, polymethyl methacrylate (manufactured by Polymer Laboratories) having a known molecular weight was used.

(3) Synthesis of temperature-responsive block copolymer Add 0.65 g (5 mmol) of 2-methoxyethyl acrylate (MEA) to a 100 mL two-necked flask, and add 31.8 mg (100 μmol) of cyanomethyldodecyltrithiocarbonate and azobis. 1.6 mg (10 μmol) of isobutyronitrile and 10 mL of tert-butanol were added, and after substitution with argon gas, the mixture was heated and stirred at 62 ° C. for 24 hours.

After the first heating and stirring, 3.85 g (30 mmol) of n-butyl acrylate (BA) was further added, 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butanol were further added, and after substitution with argon gas, The mixture was heated and stirred at 62 ° C. for 48 hours.

After the second heating and stirring, 7.36 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST = 32 ° C.) was further added, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 35 mL of tert-butanol were further added. After substitution with argon gas, the mixture was heated and stirred at 62 ° C. for 48 hours.

After the third heating and stirring, the reaction solution was reprecipitated and purified with water, and dried under reduced pressure to obtain a pale yellow solid. The obtained yellow solid was dissolved in chloroform, magnesium sulfate was added, and the mixture was stirred for 1 hour, and the filtrate was recovered. The recovered filtrate was reprecipitated and purified with heptane to obtain 7.82 g of a temperature-responsive block copolymer poly (MEA-BA-IPAAm). The composition of the obtained block copolymer was MEA / BA / IPAAm = 5/30/65 (mol%), Mn = 11.0 × 10 ⁴ , and Mw / Mn = 1.5.

(4) Preparation of temperature-responsive culture equipment A coating agent consisting of 0.3 wt% temperature-responsive block copolymer / 2-butanol solution was prepared. A temperature-responsive culture equipment 1 was obtained by dropping 270 μL of a coating agent onto a φ100 mm IWAKI tissue culture dish and spin-coating under the conditions of 3000 rpm for 1 minute. When the contact angle of air bubbles in the water of the temperature-responsive culture equipment 1 was confirmed in increments of 1 ° C. and the LCST was analyzed, the LCST of the temperature-responsive culture equipment 1 was 30 ° C.

[Example 1]
Bone marrow-derived human mesenchymal stem cells (Product Code: PT-2501, Lot Number: 0000603525) seeded in a temperature-responsive culture device 1 at 1.0 × 10 ⁵ cells / dish and used as a medium. Was added, and the cells were cultured at 37 ° C. and a CO ₂ concentration of 5%. As the medium, Dulbecco Fort's modified Eagle's minimum essential medium (10vol% FBS / DMEM) containing 10 vol% of fetal bovine serum (from Colombia) was used. The cells were cultured for 5 days, and it was confirmed that the cells had grown to the subconfluent.

A silicon tube with an inner diameter of 1.15 mm and a length of 100 cm was installed in a microtube pump (MP-2000 manufactured by Tokyo Rika Kikai). A part (30 cm) of the central part of the silicon tube was immersed in a water tank cooled to 5 ° C. with a low temperature circulator. Both ends of the silicon tube were immersed in the medium of the temperature-responsive culture device 1 in which cell proliferation was confirmed to the subconfluent, and the tube pump was operated at 3 mL / min for 4 minutes. In this system (see FIG. 1), the volume of a 100 cm silicone tube is about 1 mL, and 1 mL of 10 mL of medium circulates in the silicone tube while being cooled. Therefore, 9 mL of the medium is present in the incubator 1 during the pump operation. After 4 minutes of operation, 1 mL of medium remains in the silicon tube, so raise the liquid feed inlet side of the silicon tube into the air layer, and use the medium remaining in the silicon tube as the temperature-responsive culture equipment 1. Discharged. Further, when the temperature-responsive culture equipment 1 was allowed to stand for 10 minutes, cell detachment proceeded. A cell suspension of cells exfoliated by pipetting was prepared. The number of collected cells was measured with a hemocytometer and found to be 4.1 × ¹⁰⁵ .

The cell suspension was seeded on four new temperature-responsive culture equipment 1 at 1.0 × 10 ⁵ cells / dish, 8 mL of the medium was added, and the cells were cultured at 37 ° C. and a CO ₂ concentration of 5%. Incubate for 5 days and cell proliferation to subconfluent. From the above, it was confirmed that subculture is possible.

As described above, according to the present invention, it is possible to provide a method for culturing adherent cells using a temperature-responsive incubator, which does not require the use of a large-scale cooling device and can be automated. By utilizing the present invention, a wide range of cells including animal cells including human cells, plant cells, and microbial cells can be efficiently subcultured. Therefore, the present invention is a basic study using cells. It can be used in a wide range of industries including medical treatment (for example, regenerative medicine), agriculture (for example, production of useful agricultural products), and industry (for example, substance production using microorganisms).

Claims (6)

It is a method of culturing adherent cells.
(A) A step of culturing adherent cells in a temperature-responsive culture device,
(B) A step of recovering the medium from the culture equipment,
(C) A step of cooling the recovered medium, and (d) a step of adding the cooled medium to the incubator.
How to include. The method according to claim 1, wherein the adherent cells are proliferated above the subconfluent in the step (a). The method according to claim 1 or 2, wherein a liquid feed pump is used to recover the medium in the step (b). The method according to any one of claims 1 to 3, wherein a water-cooled cooler is used to cool the medium in the step (c). The method according to any one of claims 1 to 4, wherein a liquid feed pump is used for adding the cooled medium in the step (d). The method according to any one of claims 1 to 5, which is a closed system.

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