JP2021065186A - Apparatus and methods for statically culturing adherent cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for statically culturing adhesive cells using beads having a surface to which adhesive cells can adhere. SOLUTION: The cell culture apparatus includes a culture unit (2A) for statically culturing adhesive cells and a control unit for controlling the operation of the culture unit (2A), and the culture unit (2A) is a culture tank. (21), a first material supply unit (22) that supplies the first material to the culture tank (21), a second material supply unit (23) that supplies the second material to the culture tank (21), and culture. The tank (21) is provided with a culture solution supply / drainage section (24) for performing a culture solution supply process and / or a culture solution discharge process, and the first material contains a culture solution and adhesive cells and is undergoing static culture. A cell culture apparatus that controls the operation of the culture unit (2A) so that the culture solution supply process and / or the culture solution discharge process is performed by the culture solution supply / discharge unit (24) while maintaining the bead deposits. .. [Selection diagram] Fig. 2

Description

The present disclosure relates to devices and methods for statically culturing adherent cells.

Since adhesive cells generally adhere to a solid surface and proliferate, the growth efficiency is extremely low simply by suspending them in a culture medium. Therefore, culturing adhesive cells requires a solid surface that serves as a scaffold for the adhesive cells to adhere and proliferate. As such a solid surface, for example, an inner wall surface of a culture tank, a surface of a microcarrier, or the like is used. As the microcarrier, for example, beads having a diameter of several micrometers to several hundreds of micrometers are used.

The cell yield that can be achieved by culturing depends on the area of the solid surface to which the adhesive cells can adhere. Therefore, in order to increase the cell yield, it is necessary to increase the area of the solid surface to which the adhesive cells can adhere.

When the inner wall surface of the culture tank is used as a scaffold for the adhesive cells to adhere and proliferate, it is necessary to increase the size of the culture tank in order to increase the cell yield. On the other hand, when the surface of the microcarriers is used as a scaffold for the adhesive cells to adhere and proliferate, it is not necessary to increase the size of the culture tank, and the number of microcarriers may be increased. Therefore, the use of microcarriers is advantageous from the viewpoint of increasing the area of the solid surface to which the adhesive cells can adhere, per unit volume of the culture medium or per unit bottom area of the culture tank.

When adherent cells are cultured using microcarriers, the adherent cells adhered to the surface of the microcarriers settle in the culture medium by gravity and deposit on the bottom of the culture tank. When the adhesive cells are deposited on the bottom of the culture tank together with the microcarriers for a long time, the supply of substances necessary for the vital activity of the adhesive cells is cut off, and the waste products discharged from the adhesive cells are discharged around the adhesive cells. It stays in. Such nutrient depletion, accumulation of waste products, and the like cause a decrease in cell proliferation (cell productivity). Therefore, in order to prevent the adhesive cells from accumulating on the bottom of the culture tank together with the microcarriers for a long time, the culture solution is stirred, and the adhesive cells adhered to the surface of the microcarriers float in the culture solution. (For example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2011-09217

The present disclosure provides a technique for statically culturing adherent cells using beads having a surface to which the adherent cells can adhere.

One aspect of the present disclosure is a cell culture apparatus including a culture unit for statically culturing adhesive cells and a control unit for controlling the operation of the culture unit.
The culture section
Incubator and
A first material supply unit that supplies the first material to the culture tank,
A second material supply unit that supplies the second material to the culture tank,
A culture solution supply / discharge unit that performs a culture solution supply process and / or a culture solution discharge process to the culture tank.
With
The first material contains a culture medium and adhesive cells.
The second material comprises beads having a diameter of 0.7 mm or more having a surface to which the adhesive cells can adhere.
An opening is formed in the bottom wall of the culture tank.
The culture solution supply / discharge unit performs the culture solution supply process and / or the culture solution discharge process through the opening.
In the control unit, the first material and the second material are supplied to the culture tank by the first material supply unit and the second material supply unit, and then, in the culture tank, the adhesive cells are subjected to. The culture solution was statically cultured in a state of being adhered to the bead deposit containing beads having a diameter of 0.7 mm or more, and the culture solution was supplied by the culture solution supply / discharge unit while the bead deposit was maintained during the static culture. The present invention relates to the cell culture apparatus that controls the operation of the culture unit so that the treatment and / or the culture solution discharge treatment is performed.

The present disclosure provides a technique for statically culturing adherent cells using beads having a surface to which the adherent cells can adhere.

FIG. 1 is a schematic view for explaining the configuration of the cell culture apparatus according to the first aspect and the second aspect of the present disclosure. FIG. 2 is a partial cross-sectional view for explaining the configuration of the culture unit included in the cell culture apparatus according to the first aspect of the present disclosure. FIG. 3 is a partial cross-sectional view for explaining the configuration of the culture unit included in the cell culture apparatus according to the second aspect of the present disclosure. FIG. 4 is an enlarged view of a region represented by reference numeral R2 in FIG. FIG. 5 is a partial cross-sectional view for explaining the cell culture method carried out by the cell culture apparatus according to the first aspect or the second aspect of the present disclosure. FIG. 6 is an enlarged view of a region represented by reference numeral R in FIG. FIG. 7 is a diagram showing the relationship between the number of cells and the lactic acid concentration measured in Reference Example 1.

Hereinafter, embodiments of the present disclosure will be described with reference to the drawings.

The present disclosure relates to devices and methods for statically culturing adherent cells. Specifically, the present disclosure relates to an apparatus and method for statically culturing adherent cells using a bead deposit containing beads having a diameter of 0.7 mm or more having a surface to which the adherent cells can adhere.

In the present disclosure, "static culture" means culturing adherent cells under conditions in which bead deposits can be maintained in the process of culturing adherent cells. When the culture solution is agitated using a stirring mechanism such as a stirrer or a stirring blade, the bead deposit cannot be maintained. Therefore, in the step of culturing the adhesive cells, the culture solution is not stirred using a stirring mechanism such as a stirrer or a stirring blade. However, "static culture" does not mean that the culture solution is always in a stationary state in the process of culturing the adhesive cells, and in the present disclosure, the culture solution supply / drainage treatment is performed during the static culture. Be told. The “culture solution supply / discharge treatment” means one or both of the culture solution supply process (the culture solution supply process to the culture tank) and the culture solution discharge process (the culture solution discharge process from the culture tank). The culture solution feeding / discharging treatment in the process of culturing the adhesive cells is performed so that the bead deposits are maintained. Further, "static culture" does not mean that the culture tank is not provided with a stirring mechanism such as a stirrer and a stirring blade. The culture tank may be provided with a stirring mechanism such as a stirrer or a stirring blade, and a step performed after the step of culturing the adhesive cells (for example, a step of peeling the adhesive cells from the beads, a step of peeling the adhesive cells from the beads Since it is no longer necessary to maintain the bead deposits in the step of collecting the adherent cells, etc.), the culture solution may be agitated using a stirring mechanism such as a stirrer or a stirring blade.

In the present disclosure, static culture of adherent cells is carried out in a state where the adherent cells are adhered to a bead deposit. In the present disclosure, the bead deposit used for static culture of adherent cells includes beads having a diameter of 0.7 mm or more having a surface to which the adherent cells can adhere. Since the bead deposit used for static culture of adherent cells contains beads having a diameter of 0.7 mm or more, the bead deposit used for static culture of adhesive cells has a diameter of 0.7 mm or more. Decreased cell proliferation (cell productivity) that occurs when beads are not included (for example, cells caused by blocking the supply of substances necessary for the vital activity of adhesive cells, retention of waste products discharged from adhesive cells, etc. It is possible to prevent a decrease in proliferation (cell productivity). Further, by adjusting the amount (number) of beads having a diameter of 0.7 mm or more, the area of the solid surface to which the adhesive cells can adhere can be adjusted, thereby adjusting the cell yield that can be achieved by culturing. can do. Further, by using beads having a diameter of 0.7 mm or more, the beads and the adhesive cells can be easily separated. Therefore, according to the apparatus and method of the present disclosure, the productivity of adherent cells by static culture can be improved.

Hereinafter, the cell culture device 1A according to the first aspect of the present disclosure and the cell culture device 1B according to the second aspect of the present disclosure will be described with reference to the drawings. In the drawings relating to the cell culture apparatus 1A and the cell culture apparatus 1B, the same members or parts are represented by the same reference numerals.

<Structure of cell culture device>
The configurations of the cell culture devices 1A and 1B will be described with reference to FIG. FIG. 1 is a schematic view showing the configurations of cell culture devices 1A and 1B.

As shown in FIG. 1, the cell culture device 1A includes a culture unit 2A for statically culturing adhesive cells and a control unit 3 for controlling the operation of the culture unit 2, and the cell culture device 1B includes adhesive cells. A culture unit 2B for statically culturing and a control unit 3 for controlling the operation of the culture unit 2 are provided.

The culture units 2A and 2B perform various treatments related to static culture of adhesive cells. In addition to the static culture treatment of the adhesive cells, the culture units 2A and 2B were subjected to various treatments (for example, a peeling treatment of the adhesive cells from the beads and a peeling from the beads) performed after the static culture treatment of the adhesive cells. (Recovery treatment of adhesive cells, etc.) may be performed.

The control unit 3 is, for example, a computer, and includes a main control unit and a storage unit. The main control unit is, for example, a CPU (Central Processing Unit), and controls the operations of the culture units 2A and 2B by reading and executing a program stored in the storage unit. The storage unit is composed of storage devices such as a RAM (Random Access Memory), a ROM (Read Only Memory), and a hard disk, and stores programs that control various processes executed in the culture units 2A and 2B. The program may be recorded on a storage medium that can be read by a computer, or may be installed in the storage unit from the storage medium. Examples of the storage medium that can be read by a computer include a hard disk (HD), a flexible disk (FD), a compact disk (CD), a magnet optical disk (MO), a memory card, and the like. The recording medium records, for example, a program in which the computer controls the operation of the cell culture device 1 to execute the cell culture method described later when executed by a computer for controlling the operation of the cell culture device 1. To.

<Structure of culture section>
The configurations of the culture units 2A and 2B will be described with reference to FIGS. 2 to 4. FIG. 2 is a partial cross-sectional view showing the configuration of the culture unit 2A, FIG. 3 is a partial cross-sectional view showing the configuration of the culture unit 2B, and FIG. 4 is a partial cross-sectional view of the region represented by reference numeral R2 in FIG. It is an enlarged view. In FIGS. 2 and 3, the Z direction represents the direction of gravity, and the expressions "upper", "upper" and "upper" used in the present specification mean the upper, upper and upper parts in the Z direction. To do.

As shown in FIG. 2, the culture unit 2A includes a culture tank 21, a first material supply unit 22 that supplies the first material M1 to the culture tank 21, and a second material that supplies the second material M2 to the culture tank 21. A supply unit 23 is provided. As shown in FIG. 3, the culture unit 2B includes a culture tank 21 and a first material supply unit 22 that supplies the first material M1 to the culture tank 21. Note that, unlike the culture unit 2A, the culture unit 2B does not include the second material supply unit 23 that supplies the second material M2 to the culture tank 21.

As shown in FIGS. 2 and 3, the culture tank 21 has a culture space S in which the adhesive cells are statically cultured. As shown in FIGS. 2 and 3, in the culture tank 21, the bottom wall portion 211, the peripheral wall portion 212 rising from the peripheral edge of the bottom wall portion 211, and the upper wall portion 213 that seals the upper opening of the peripheral wall portion 212 are sealed. The culture space S is surrounded by a bottom wall portion 211, a peripheral wall portion 212, and an upper wall portion 213. Therefore, the culture space S of the culture tank 21 is suitable for closed system culture. The volume of the culture space S can be appropriately adjusted in consideration of the culture scale to be realized and the like. The volume of the culture space S is preferably 1 mL or more and 10,000 mL or less, more preferably 5 mL or more and 1,000 mL or less, and even more preferably 10 mL or more and 100 mL or less. When it is not necessary to carry out closed system culture, the upper wall portion 213 may be omitted. Hereinafter, the wall surface of the culture tank 21 on the culture space S side is referred to as an inner wall surface of the culture tank 21, the wall surface of the bottom wall portion 211 on the culture space S side, the wall surface of the peripheral wall portion 212 on the culture space S side, and the upper wall portion 213. The wall surfaces on the culture space S side of the above are referred to as an inner wall surface of the bottom wall portion 211, an inner wall surface of the peripheral wall portion 212, and an inner wall surface of the upper wall portion 213, respectively.

The inner wall surface of the bottom wall portion 211 of the culture tank 21 may be a flat surface or a curved surface, but is usually a flat surface. The shape of the inner wall surface of the bottom wall portion 211 of the culture tank 21 is not particularly limited. Examples of the shape of the inner wall surface of the bottom wall portion 211 of the culture tank 21 include a circular shape, an elliptical shape, and a rectangular shape.

As shown in FIGS. 2 and 3, the shape of the peripheral wall portion 212 of the culture tank 21 is preferably a shape that is inclined toward the bottom wall portion 211 and is tapered, that is, a tapered shape. By making the peripheral wall portion 212 of the culture tank 21 tapered, the formation of turbulent flow during the supply and discharge of the culture solution can be suppressed, and the efficiency of supply and discharge of the culture solution can be improved. Further, by making the peripheral wall portion 212 of the culture tank 21 tapered, it is possible to prevent air bubbles from remaining at the portion where the peripheral wall portion 212 and the bottom wall portion 211 are in contact with each other, and the culture space S of the culture tank 21 can be prevented from remaining. The whole can be completely filled with the culture medium.

When the peripheral wall portion 212 is tapered, the taper angle (the angle formed by the inner wall surface of the bottom wall portion 211 and the inner wall surface of the peripheral wall portion 212) is preferably larger than 90 degrees and 150 degrees or less, more preferably 90 degrees or more. It is largely 135 degrees or less, and even more preferably more than 90 degrees and 105 degrees or less. By setting the taper angle within the above range, the formation of turbulent flow at the time of feeding and discharging the culture solution can be suppressed more effectively. Further, since the area of the bottom wall portion 212 can be designed to be smaller than the maximum area of the culture space S in the horizontal direction, the cells seeded in the culture space S can be placed in the culture space S from the opening of the bottom wall portion 212. You can prevent them from going out.

The inner wall surface of the culture tank 21 (particularly, the inner wall surface of the bottom wall portion 211 and the inner wall surface of the peripheral wall portion 212) is preferably cell-non-adhesive. By making the inner wall surface of the culture tank 21 non-adhesive to cells, the main solid surface to which the adhesive cells adhere can be the bead surface. Thereby, the area of the solid surface (and thus the cell yield) to which the adhesive cells adhere and proliferate can be controlled and controlled by the number of beads.

The cell non-adhesiveness of the inner wall surface of the culture tank 21 can be imparted by applying a cell non-adhesive coating to the inner wall surface of the culture tank 21. Examples of the cell non-adhesive coating include celluloses such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose; polyethylene oxide; carboxyvinyl polymer; polyvinylpyrrolidone; polyethylene glycol; polylactic acid; polyacrylamide, and poly. Polypolymers such as N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, dextran; phospholipid polymers; coatings with derivatives thereof and the like. Further, the cell non-adhesiveness of the inner wall surface of the culture tank 21 can be imparted by forming the culture tank 21 itself with a cell non-adhesive material. Examples of the cell non-adhesive material include synthetic resins (for example, polystyrene, polycarbonate, tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA)) and polytetrafluoroethylene (for example, polystyrene, polycarbonate, tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer), which have not been subjected to surface charge by plasma treatment or the like. PTFE), etc.).

As shown in FIGS. 2 and 3, an opening 214 leading to the culture space S is formed in the bottom wall portion 211 of the culture tank 21. As shown in FIGS. 2 and 3, a pipe 240 is connected to the opening 214 formed in the bottom wall portion 211 of the culture tank 21, and the pipe 240 is connected to the culture tank 21 through the opening 214 and the pipe 240. , First material supply / discharge treatment by the first material supply unit 22 (one or both of supply of the first material M1 to the culture tank 21 and discharge of the first material M1 from the culture tank 21) and / or supply / discharge of the culture solution. The culture solution supply / discharge treatment (discharge of the culture solution from the culture tank 21 and supply of the culture solution to the culture tank 21 or both) can be performed by the unit 24. When bead deposits are present in the culture space S of the culture tank 21, the first material supply / discharge treatment and / or the culture solution supply / discharge treatment is performed through the opening 214 formed in the bottom wall portion 211 of the culture tank 21. By doing so, it is easy to maintain the bead deposit during the first material supply / discharge treatment and / or the culture solution supply / discharge treatment. An opening for performing the first material supply / discharge treatment and / or the culture solution supply / discharge treatment on the culture tank 21 may be formed in the peripheral wall portion 212 or the upper wall portion 213. The number of openings for performing the first material supply / discharge treatment and / or the culture solution supply / discharge treatment on the culture tank 21 may be 2 or more. An opening for performing the first material discharge treatment and / or a culture solution discharge treatment for the culture tank 21 and an opening for performing the first material supply treatment and / or the culture solution supply treatment for the culture tank 21. And may be provided separately.

As shown in FIGS. 2 and 3, the pipe 240 is provided with a flow path switching portion 27. The flow path switching portion 27 is a mechanism provided as needed and can be omitted. The present disclosure also includes an embodiment in which the flow path switching unit 27 is omitted.

The flow path switching unit 27 includes, for example, a flow path switching valve as a flow path switching mechanism. The flow path switching unit 27 switches the pipe connected to the pipe 240 to the pipe 240, a pipe 242a for discharging the culture solution from the desired pipe (for example, the culture space S of the culture tank 21), and the culture. The pipe 242b for supplying the culture solution to the culture space S of the tank 21 or the pipe 222) for supplying the first material M1 to the culture space S of the culture tank 21 is connected.

As shown in FIGS. 2 and 3, the bottom wall portion 211 of the culture tank 21 is provided with a filter F that covers the opening 214. The filter F is a filter for preventing the outflow of beads from the culture space S of the culture tank 21. The opening of the filter F is adjusted so that the liquid can pass through but the beads cannot pass through. The opening of the filter F can be appropriately adjusted according to the diameter of the beads, but is preferably not more than half the diameter of the beads. The solid-liquid separation by the filter F is performed by, for example, a dead-end filtration method. The filter F has a structure provided as needed and can be omitted. The present disclosure also includes embodiments in which the filter F is omitted.

The culture tank 21 is held by a holding portion (not shown) so that static culture can be performed. The environment of the culture space S of the culture tank 21 is maintained in an environment suitable for culturing adhesive cells by an environment adjustment unit (not shown). Environment adjustment unit includes, for example, temperature and humidity adjustment unit for adjusting the temperature and humidity of the culture space S of the culture tank 21, the CO ₂ concentration adjusting unit for adjusting the CO ₂ concentration in the culture space S of the culture tank 21. The temperature / humidity adjusting unit adjusts the temperature and humidity of the culture space S of the culture tank 21 to, for example, 37 ° C. and 95% or more, respectively. CO ₂ concentration adjusting unit adjusts the CO ₂ concentration in the incubation space S of the culture tank 21, for example 5%. The environment adjustment unit can be configured in the same manner as a known incubator used for cell culture.

As shown in FIG. 2, the culture space S of the culture tank 21 in the culture unit 2A is empty, and a gas such as air is present. On the other hand, as shown in FIG. 3, the bead deposit L is housed in the culture space S of the culture tank 21 in the culture unit 2B. As shown in FIG. 4, the bead deposit L is _{formed by stacking a large number of beads B 0} , and one bead B _{0 and another bead B 0} existing adjacent to the certain bead B _0. A gas such as air exists in the gap existing between the beads.

The shape of the beads constituting the bead deposit L is substantially spherical. The diameter of the beads is measured as follows. Observe the beads using a phase-contrast microscope, quantify the area of the beads taken from directly above, and calculate the bead diameter from the quantified area values, assuming the beads are true spheres. To do.

The bead deposit L contains beads having a diameter of 0.7 mm or more. The beads having a diameter of 0.7 mm or more are preferably beads having a diameter of 1.5 mm or more, and more preferably beads having a diameter of 3.0 mm or more. The upper limit of the bead diameter is not particularly limited, but if the bead diameter is too large, when the adhesive cells are adhered to the bead deposit L and statically cultured, the adhesive cells adhere to the surface of the beads. After growing, it becomes difficult to move to the surface of another bead adjacent to the bead and further grow. Bead diameter from the perspective of allowing adherent cells to migrate from the surface of one bead to the surface of another bead adjacent to the bead within a few days (eg, about 3 days). Is preferably 5.0 mm or less.

The bead deposit L may be composed of two or more types of beads having different diameters. The beads constituting the bead deposit L may contain beads having a diameter of less than 0.7 mm in addition to beads having a diameter of 0.7 mm or more, but do not include beads having a diameter of less than 0.7 mm. Is preferable. The amount (number) of beads having a diameter of less than 0.7 mm is preferably 10% or less, more preferably 0.1% or less, still more preferably, based on the total amount (total number) of beads constituting the bead deposit L. Is 0.001% or less, most preferably zero.

The specific gravity of the beads constituting the bead deposit L is not particularly limited as long as it is larger than the specific gravity of the culture solution (that is, the beads can settle in the culture solution by gravity), but the specific gravity of the beads (37 ° C.) is not particularly limited. It is preferably 1.01 or more and 3.00 or less, and more preferably 1.04 or more and 3.00 or less. When the specific gravity of the beads is smaller than the specific gravity of the culture solution, the beads float and it becomes difficult to control the position of the beads, and cell detachment from the beads is likely to occur due to contact between the beads. On the other hand, if the specific gravity of the beads is too large, the total weight of the culture system becomes large and it becomes difficult to handle. From the viewpoint of handling, the total weight of the culture system is preferably small, and from the viewpoint of reducing the total weight of the culture system, the specific gravity of the beads is preferably as small as possible. Further, from the viewpoint of improving the cell recovery efficiency at the time of cell recovery, it is preferable that the beads are easy to float, and from the viewpoint that the beads are easy to float, it is preferable that the specific gravity of the beads is as small as possible.

The beads that make up the bead deposit L have a surface to which adherent cells can adhere. The beads can be formed of, for example, a synthetic resin such as a thermoplastic resin or a thermosetting resin. Examples of the thermoplastic resin include styrene resin, (meth) acrylic resin, vinyl acetate resin, vinyl ether resin, halogen-containing resin, alicyclic olefin resin, polycarbonate resin, polyester resin, and polyamide resin. , Cellulose derivatives and the like. Examples of the thermosetting resin include silicone-based resins, rubbers, elastomers, and the like. The surface of the resin may be surface-treated in order to improve cell adhesion. Examples of the surface treatment include hydrophilization treatment by oxygen plasma irradiation, addition of surface positive charge by ammonia plasma irradiation, and the like. In order to further improve cell adhesion, the resin surface may be coated with a material to which adhesive cells can adhere to form a coating layer. Examples of materials to which adherent cells can adhere include cell adhesion factors such as ECM (Extracellular Matrix), hydrophilic substances such as collagen, gelatin, fibronectin, laminin, polylysine, thrombosponectin, and vitronectin.

The thickness T _{1 of the} bead deposit L (the length of the bead deposit L in the direction of gravity Z) is not particularly limited. The larger the thickness T ₁ of the bead deposit L, adherent cells increases the area of culturable solid surface. Therefore, the thickness T ₁ of the bead deposit L is preferably 3 times or more, more preferably 20 times or more, still more preferably 40 times or more, still more preferably 100 times the diameter of the beads constituting the bead deposit L. More than double. On the other hand, _{if the thickness T 1} of the bead deposit L is too large, the amount of the culture solution required for culturing the adhesive cells increases, the treatment time with the cell exfoliating solution P increases, and the like, and the control of a series of steps becomes complicated. To become. Therefore, the thickness T ₁ of the bead deposit L (the length of the bead deposit L in the direction of gravity Z) is preferably 1000 times or less, more preferably 200 times or less, and further preferably the diameter of the beads constituting the bead deposit L. More preferably, it is 120 times or less. In the present disclosure, _{the value obtained by dividing the thickness T 1} of the bead deposit L by the diameter of the beads constituting the bead deposit L (thickness T ₁ of the bead deposit L / diameter of the beads) is divided by the bead deposit L. It is called the number of layers.

If the thickness of the bead deposit L is not uniform, the maximum thickness of the bead deposit L is used as _{the thickness T 1 of the bead deposit L for calculating the number of layers of the bead deposit L.}

When the bead deposit L is composed of two or more types of beads having different diameters, the diameter of the bead having the largest total surface area among the two or more types of beads calculates the number of layers of the bead deposit L. Used as the diameter of the beads for. When there are two or more types of beads having the largest total surface area, the diameter of the bead having the largest diameter among the two or more types of beads is used as the diameter of the beads for calculating the number of layers of the bead deposit L. Ru. For example, bead deposit L is a _{N 1} beads _{B 1} having a surface area SA _1, when configured for the _{N 2} beads _{B 2} having a surface area SA _2, the total surface area of the bead _{B 1} = SA The total surface area of ₁ × N ₁ and beads B _{2 = SA 2} × N ₂ . When the total surface area of the bead B ₁ is larger than the total surface area of the bead B _2, when the diameter of the bead B ₁ is the total surface area of the bead B ₂ is larger than the total surface area of the beads B ₁ represents a bead B ₂ Is used as the bead diameter for calculating the number of layers of bead deposit L. When the total surface area of the bead B _{1 and} the total surface area of the bead B ₂ are equal _{, the diameter of the bead having the largest diameter among the bead B 1} and the bead B ₂ is used to calculate the number of layers of the bead deposit L. Used as the bead diameter of.

The first material supply unit 22 supplies the first material M1 to the culture space S of the culture tank 21. The first material M1 contains a culture medium and adhesive cells. The first material M1 is a mixture of a culture medium and adhesive cells (cell suspension), and the adhesive cells are dispersed in the culture medium. As shown in FIGS. 2 and 3, the first material supply unit 22 puts the tank 221 in which the first material M1 is stored and the first material M1 stored in the tank 221 into the culture space S of the culture tank 21. It is provided with a supply pipe 222 for supplying to. As shown in FIGS. 2 and 3, the supply pipe 222 connects the tank 221 and the flow path switching portion 27, and the first material M1 stored in the tank 221 has a pipe 240 and an opening 214. It is supplied to the culture space S of the culture tank 21 via. As shown in FIGS. 2 and 3, the supply pipe 222 is provided with a flow rate adjusting unit 223 such as a valve and a pump, and the flow rate of the first material M1 supplied to the culture space S of the culture tank 21. The flow rate and the like (supply amount of culture solution, supply rate of culture solution, supply amount of adhesive cells, supply rate of adhesive cells, etc.) are adjusted by the flow rate adjusting unit 223. In the present embodiment, the first material supply unit 22 supplies the first material M1 to the culture space S of the culture tank 21 through the opening 214 of the culture tank 21, but the first material M1 by the first material supply unit 22 The supply form of is not limited to this. For example, the supply pipe 222 may extend from the tank 221 through the upper wall portion 213, the peripheral wall portion 212, or the bottom wall portion 211 of the culture tank 21 to the culture space S of the culture tank 21. In this case, the first material M1 stored in the tank 221 is supplied to the culture space S of the culture tank 21 from the tip of the supply pipe 222.

The culture solution contained in the first material M1 is not particularly limited as long as the adhesive cells can be cultured, and can be appropriately selected. Examples of the culture medium include a cell culture basic medium, a differentiation medium, a medium dedicated to primary culture, and the like. Specifically, Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI1640, Ham F-12, MCDB medium, Williams medium E, and a mixture thereof. Examples include a medium. The culture medium can be used as long as it is a medium containing components necessary for the proliferation and differentiation of adhesive cells. Serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts and the like may be added to the culture broth depending on the purpose of culturing (cell proliferation, differentiation, etc.). When the lactic acid concentration in the culture solution is measured and the number of cells is estimated based on the measured lactic acid concentration, it is preferable that the culture solution does not contain lactic acid.

The adhesive cells contained in the first material M1 are not particularly limited as long as they can adhere to the solid surface and proliferate, and can be appropriately selected. In the present disclosure, the main solid surface that serves as a scaffold for adhesive cells to adhere and proliferate is the surface of the beads that make up the bead deposit L. Examples of adhesive cells include pluripotent stem cells, stem cells, progenitor cells, somatic cells, germ cells and the like. The first material M1 may contain one type of adhesive cells, or may contain two or more types of adhesive cells.

Pluripotent stem cells mean cells capable of differentiating into cells from any of the three germ layers. Examples of pluripotent stem cells include embryonic stem cells (ES cells), inducible pluripotent stem cells (iPS cells), Muse cells (Multilineage-differentiating Stress Enduring Cell), embryonic tumor cells (EC cells), and embryonic stem cells. Examples thereof include embryonic stem cells (EG cells).

Stem cells are defined as cells that have the ability to divide to produce the same cells as themselves and to differentiate into different types of cells, allowing them to proliferate endlessly. Examples of stem cells include mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, neural stem cells, muscle stem cells, germline stem cells (egg progenitor cells and spermatogenic cells) and the like.

The animal from which the adhesive cell is derived is not particularly limited, and may be a vertebrate or an invertebrate. Examples of vertebrates include mammals, amphibians, reptiles, amphibians, fish and the like. Examples of invertebrates include insects, crustaceans, mollusks, protozoa and the like. The animal from which the adherent cells are derived is preferably a vertebrate, more preferably a mammal. Examples of mammals include humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, etc. Among these, humans are preferable.

Adhesive cells may form tissue. Examples of the tissue include cartilage tissue, bone tissue, muscle tissue, corneal tissue, vascular tissue and the like. The tissue may be a tissue separated from a living body or a tissue differentiated from stem cells.

The concentration of adherent cells contained in the first material M1 is determined by the type of adherent cells used, the amount of the first material M1 supplied to the culture tank 21, the culture conditions used, and the bead deposits in the culture tank 21. It can be adjusted according to the total surface area of L and the like. The concentration of adhesive cells contained in the first material M1 is preferably adjusted so that the number of cells per unit surface area of the bead deposit L is within a predetermined range. For example, when mesenchymal stem cells are used, the number of cells per unit surface area of the bead deposit L is preferably 10 cells / cm ² or more and 20,000 cells / cm ² or less, and more preferably 100 cells / cm ² or more. It is preferably adjusted to 10,000 cells / cm ² or less, and even more preferably 1,000 cells / cm ² or more and 7,000 cells / cm ^{2 or less.} The total surface area of the bead deposit L can be calculated from the number and diameter of the beads constituting the bead deposit L. The number of beads may be measured manually using, for example, a counting machine, or the total weight of the bead deposit L may be measured and divided by the weight per bead. The method for measuring the diameter of the beads is as described above. The method for measuring the number of beads is as follows. When the bead deposit L is composed of two or more types of beads having different diameters, the diameter of the bead having the largest total surface area among the two or more types of beads calculates the total surface area of the bead deposit L. Used as the diameter of the beads for. When two or more types of beads having the largest total surface area exist, the diameter of the bead having the largest diameter among the two or more types of beads is used as the diameter of the beads for calculating the total surface area of the bead deposit L. Ru.

The second material supply unit 23 supplies the second material M2 to the culture space S of the culture tank 21. The second material M2 is a fluid containing beads having a diameter of 0.7 mm or more having a surface to which adhesive cells can adhere, and is, for example, a bead dispersion liquid, a bead powder, or the like. As shown in FIG. 2, the second material supply unit 23 supplies the tank 231 in which the second material M2 is stored and the second material M2 stored in the tank 231 to the culture space S of the culture tank 21. It is provided with a supply pipe 232. As shown in FIG. 2, the supply pipe 232 extends from the tank 231 through the upper wall portion 213 of the culture tank 21 to the culture space S of the culture tank 21, and is stored in the tank 231. The material M2 is supplied from the tip of the supply pipe 232 to the culture space S of the culture tank 21. As shown in FIG. 2, the supply pipe 232 is provided with a flow rate adjusting unit 233 such as a valve and a pump, and the flow rate, flow rate, etc. of the second material M2 supplied to the culture space S of the culture tank 21 ( That is, the supply amount of beads, the supply speed, etc.) are adjusted by the flow rate adjusting unit 233. The supply pipe 232 may extend from the tank 231 through the peripheral wall portion 212 or the bottom wall portion 211 of the culture tank 21 to the culture space S of the culture tank 21. When the second material M2 is bead powder, it is preferable that the tank 231 is provided with a mechanism for adjusting the number of beads stored in the tank 231.

The bead dispersion liquid contains a dispersion medium and beads dispersed in the dispersion medium. As the dispersion medium contained in the bead dispersion, for example, a culture solution of adhesive cells can be used. The bead powder is a powder composed of beads.

When the second material M2 is supplied to the culture space S of the culture tank 21 by the second material supply unit 23, the bead deposit L is formed in the culture space S of the culture tank 21. The description of the bead deposit L is the same as above.

As shown in FIGS. 2 and 3, the culture unit 2 supplies and discharges the culture solution to the culture tank 21 (discharge of the culture solution from the culture tank 21 and supply of the culture solution to the culture tank 21, or both. ) Is provided. The culture solution supply / discharge unit 24 performs the culture solution supply process and / or the culture solution discharge process through the opening 214 of the culture tank 21.

As shown in FIGS. 2 and 3, the culture solution supply / discharge section 24 includes a culture solution discharge section 24a and a culture solution supply section 24b.

The culture solution discharge unit 24a performs a culture solution discharge process (discharge of the culture solution from the culture tank 21). The discharged culture medium is usually a culture medium (used culture medium) after being used for culturing adhesive cells. The discharged culture solution may be the culture solution contained in the first material M1 supplied by the culture material supply unit 22, or may be the culture solution supplied by the culture solution supply unit 24b. It may be a mixture of these. As shown in FIGS. 2 and 3, the culture solution discharge unit 24a connects the tank 241a containing the culture solution M3 discharged from the culture space S of the culture tank 21 to the tank 241a and the flow path switching unit 27. It is provided with a pipe 242a. When the pipe 240 and the pipe 242a are connected by the flow path switching portion 27, the culture solution M3 discharged from the culture space S of the culture tank 21 is housed in the tank 241a through the opening 214, the pipe 240 and the pipe 242a. To. As shown in FIGS. 2 and 3, a flow rate adjusting unit 243a such as a valve or a pump is provided in the pipe 242a, and the flow rate, flow rate, etc. of the culture solution M3 discharged from the culture space S of the culture tank 21 are provided. Is adjusted by the flow rate adjusting unit 243a.

The control unit 3 controls the operation of the culture solution discharge unit 24a so that the culture solution discharge process is performed by the culture solution discharge unit 24a while maintaining the bead deposit L. The bead deposit L can be maintained by adjusting the rate of descent of the liquid level in the culture liquid discharge treatment. From the viewpoint of maintaining the bead deposit L, the rate of descent of the liquid level in the culture solution discharge treatment is preferably 30 mm / min or less, more preferably 10 mm / min or less, and even more preferably 1 mm / min or less.

The culture solution supply unit 24b performs a culture solution supply process (supply of the culture solution M4 to the culture tank 21). The culture solution M4 supplied to the culture tank 21 is usually a culture solution (fresh culture solution) that is not used for culturing adhesive cells. As shown in FIGS. 2 and 3, the culture solution supply unit 24b includes a tank 241b in which the culture solution M4 is stored, and a pipe 242b that connects the tank 241b and the flow path switching unit 27. When the pipe 240 and the pipe 242b are connected by the flow path switching portion 27, the culture solution M4 stored in the tank 241b is supplied to the culture space S of the culture tank 21 through the pipe 242b, the pipe 240 and the opening 214. Will be done. As shown in FIGS. 2 and 3, a flow rate adjusting unit 243b such as a valve or a pump is provided in the pipe 242b, and the flow rate, flow rate, etc. of the culture solution M4 supplied to the culture space S of the culture tank 21 are provided. Is adjusted by the flow rate adjusting unit 243b. As the culture solution M4 stored in the tank 241b, the same culture solution as the culture solution contained in the first material M1 can be used.

The culture solution M4 supplied to the culture tank 21 by the culture solution supply unit 24b is supplied by the culture solution (for example, the first material supply unit 22) after being used for culturing adhesive cells in the culture space S of the culture tank 21. The culture solution contained in the first material M1 obtained) may be used. In this case, the culture solution supply unit 24b temporarily collects a part of the culture solution from the culture tank 21 and stores it in the tank 241b, and then supplies it to the culture space S of the culture tank 21 again. When a part of the culture solution is temporarily collected from the culture tank 21, the flow rate, flow rate, etc. of the collected culture solution (culture solution supplied to the tank 241b) are adjusted by the flow rate adjusting unit 243b. When a part of the culture solution is temporarily collected from the culture tank 21, it is preferable to adjust the amount of the culture solution collected so that the bead deposit L is not exposed. By temporarily collecting the culture solution from the culture space S of the culture tank 21 and supplying it to the culture space S of the culture tank 21 again, the culture solution in the culture tank 21 can be agitated.

The control unit 3 controls the operation of the culture solution supply unit 24b so that the culture solution supply process is performed by the culture solution supply unit 24b while the bead deposit L is maintained. The bead deposit L can be maintained by adjusting the rate of rise of the liquid level in the culture liquid supply treatment. From the viewpoint of maintaining the bead deposit L, the rate of rise of the liquid level in the culture solution supply treatment is preferably 30 mm / min or less, more preferably 10 mm / min or less, and even more preferably 1 mm / min or less.

As shown in FIGS. 2 and 3, the culture units 2A and 2B further include a lactic acid concentration measuring unit 25 for measuring the lactic acid concentration in the culture solution existing in the culture space S of the culture tank 21. The lactic acid concentration measuring unit 25 has a structure provided as needed and can be omitted. The present disclosure also includes an embodiment in which the lactate concentration measuring unit 25 is omitted. The culture solution for which the lactic acid concentration is measured may be the culture solution contained in the first material M1 supplied by the first material supply unit 22, or the culture solution supplied by the culture solution supply unit 24b. It may be a mixture of these. The lactate concentration of either culture is usually measured after the culture has been used to culture adherent cells.

The lactic acid concentration measuring unit 25 includes a culture solution existing in the culture space S (for example, a culture solution contained in the first material M1 supplied by the first material supply unit 22, a culture solution supplied by the culture solution supply unit 24b, and the like. Alternatively, a part of (a mixture of these) is collected, and the lactic acid concentration in a part of the collected culture solution is measured. It is preferable that a part of the culture solution is collected from the culture solution existing above the bead deposit L. It is preferable that the control unit 3 determines the continuation or termination of the static culture of the adhesive cells in the culture tank 21 based on the lactate concentration measured by the lactate concentration measuring unit 25. Lactic acid is a cell metabolite secreted from cells, and the lactic acid concentration is an index of the number of cells. That is, the number of cells can be estimated based on the lactate concentration (the higher the lactate concentration, the larger the number of cells). Therefore, for example, when the lactic acid concentration is equal to or higher than the predetermined reference value, the control unit 3 ends the static culture of the adhesive cells, and when the lactic acid concentration is less than the predetermined reference value, the control unit 3 is adhesive. Continue static culture of cells.

As shown in FIGS. 2 and 3, the culture units 2A and 2B further include a cell exfoliation solution supply unit 26 that supplies the cell exfoliation solution P that exfoliates adhesive cells from the beads to the culture space S. The cell exfoliating liquid supply unit 26 has a structure provided as needed and can be omitted. The present disclosure also includes an embodiment in which the cell exfoliating liquid supply unit 26 is omitted.

The cell stripping solution supply unit 26 supplies the cell stripping solution P to the culture tank 21. Examples of the cell exfoliating agent contained in the cell exfoliating solution P include enzymes such as trypsin, collagenase, dispase and acutase, and chelating agents such as EDTA (ethylenediaminetetraacetic acid). The solvent contained in the cell exfoliating solution P is not particularly limited as long as the survival of the adhesive cells can be maintained. Examples of the solvent include water, phosphate buffered saline (PBS), Tris buffered saline (TBS) and the like. As shown in FIGS. 2 and 3, the cell exfoliating solution supply unit 26 uses the tank 261 in which the cell exfoliating solution P is stored and the cell exfoliating solution P stored in the tank 261 in the culture space S of the culture tank 21. It is provided with a supply pipe 262 for supplying to. As shown in FIGS. 2 and 3, the supply pipe 262 extends from the tank 261 through the upper wall portion 213 of the culture tank 21 to the culture space S of the culture tank 21 and is stored in the tank 261. The cell stripping solution P is supplied from the tip of the supply tube 262 to the culture space S of the culture tank 21. As shown in FIGS. 2 and 3, the supply pipe 262 is provided with a flow rate adjusting unit 263 such as a valve and a pump, and the flow rate of the cell exfoliating liquid P supplied to the culture space S of the culture tank 21. The flow rate and the like (supply amount of cell exfoliating liquid P, supply speed, etc.) are adjusted by the flow rate adjusting unit 263. The supply pipe 262 may extend from the tank 261 through the peripheral wall portion 212 or the bottom wall portion 211 of the culture tank 21 to the culture space S of the culture tank 21. Further, the supply pipe 262 may be connected from the tank 261 to the above-mentioned flow path switching portion 27. In this case, the cell exfoliating liquid P stored in the tank 261 passes through the pipe 240 and the opening 214. Is supplied to the culture space S of the culture tank 21.

<Cell culture method>
The cell culture method according to the embodiment of the present disclosure will be described with reference to FIGS. 5 and 6. The cell culture method according to this embodiment is carried out by the cell culture device 1A or 1B. FIG. 5 is a partial cross-sectional view for explaining the cell culture method carried out by the cell culture devices 1A and 1B, and FIG. 6 is an enlarged view of a region represented by reference numeral R in FIG. In FIG. 5, a part of the cell culture devices 1A and 1B is omitted.

The cell culture method according to the embodiment of the present disclosure is
(A) A step of preparing a culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L; and (b) in the culture tank 21, the adhesive cells were adhered to the bead deposit L. It includes a step of statically culturing in a state.

The culture tank 21 is held by a holding portion (not shown) so that static culture can be performed. The environment of the culture space S of the culture tank 21 is maintained in an environment suitable for culturing adhesive cells by an environment adjustment unit (not shown). The temperature and humidity of the culture space S of the culture tank 21 are adjusted to, for example, 37 ° C. and 95% or more, respectively, by a temperature / humidity adjusting unit (not shown). _{The CO 2} concentration in the culture space S of the culture tank 21 is adjusted to, for example, 5% by _{a CO 2} concentration adjusting unit (not shown).

The step (a) is a step of preparing a culture tank 21 for accommodating the adhesive cells, the culture solution C, and the bead deposit L. The bead deposit L contains beads having a diameter of 0.7 mm or more having a surface to which adhesive cells can adhere. The description of the culture medium, the adhesive cells and the bead deposit L is the same as described above.

As shown in FIG. 5, the entire bead deposit L is immersed in the culture solution C. As shown in FIG. 6, the bead deposit L is _{formed by stacking a large number of beads B 0} , and one bead B _{0 and another bead B 0} existing adjacent to the certain bead B _0. The culture solution is present in the gap V existing between the two.

Hereinafter, a case where the step (a) is carried out using the cell culture apparatus 1A will be described.

When the step (a) is carried out using the cell culture apparatus 1A, the first material supply unit 22 supplies the first material M1 containing the culture solution and the adhesive cells to the culture space S of the culture tank 21 and at the same time. The second material supply unit 23 supplies the second material M2 to the culture space S of the culture tank 21. An additional culture solution may be supplied to the culture space S of the culture tank 21 by the culture solution supply unit 24b.

The order of supplying the first material M1 and the supply of the second material M2 is not particularly limited. For example, the second material M2 may be supplied after the supply of the first material M1, the first material M1 may be supplied after the supply of the second material M2, or the first material M1 may be supplied. And the supply of the second material M2 may be performed at the same time. When the additional culture solution is supplied by the culture solution supply unit 24b, the supply of the additional culture solution may be performed before the supply of the first material M1 or after the supply of the first material M1. It may be carried out at the same time as the supply of the first material M1, but it is usually carried out after the supply of the first material M1.

In the first embodiment, the second material M2 is bead powder, and after the supply of the second material M2, the first material M1 is supplied. The amount of second material supplied is adjusted to form the bead deposit L. The bead deposit L is immediately formed by the supply of the second material M2. The amount of the first material M1 supplied is adjusted so that the entire bead deposit L is immersed in the culture medium contained in the first material M1. It is preferable that the bead deposit L is maintained during the supply of the first material M1, but it is not essential that the bead deposit L is maintained during the supply of the first material M1.

In the first embodiment, when the bead deposit L is maintained during the supply of the first material M1, the supply of the first material M1 accommodates the adhesive cells, the culture medium C, and the bead deposit L. The culture tank 21 is prepared.

In the first embodiment, if the bead deposit L is not maintained during the supply of the first material M1, a mixture of culture medium, adhesive cells and beads is formed. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

In the second embodiment, the second material M2 is bead powder, the first material M1 is supplied after the supply of the second material M2, and the additional culture solution is supplied after the supply of the first material M1. Will be. The amount of second material supplied is adjusted to form the bead deposit L. The bead deposit L is immediately formed by the supply of the second material M2. The amount of the first material M1 supplied is such that the lower part of the bead deposit L is immersed in the culture solution contained in the first material M1 (that is, the upper part of the bead deposit L is exposed to the culture space S). ) Adjusted. The amount of the additional culture solution supplied is adjusted so that the entire bead deposit L is immersed in the culture solution (the culture solution contained in the first material M1 and the additional culture solution). That is, a part of the culture solution C is supplied so that the lower part of the bead deposit L is immersed in the part of the culture solution C (that is, the upper part of the bead deposit L is exposed to the culture space S). The rest of the culture solution C is then supplied so that the entire bead deposit L is immersed in the culture solution C (ie, the upper part of the bead deposit L is not exposed to the culture space S). The entire bead deposit L may be finally immersed in the culture solution C, and the amount of the first material M1 supplied and the amount of the additional culture solution supplied can be adjusted as appropriate. It is preferable that the bead deposit L is maintained during the supply of the first material M1 and the supply of the additional culture medium, but the bead deposit L is maintained during the supply of the first material M1 and the supply of the additional culture solution. It is not essential to be maintained.

In the second embodiment, when the bead deposit L is maintained during the supply of the first material M1 and the additional culture medium, the adhesive cells and the adhesive cells are supplied by the supply of the first material M1 and the additional culture medium. A culture tank 21 containing the culture solution C and the bead deposit L is prepared.

In the second embodiment, if the bead deposit L is not maintained during the supply of the first material M1 and the additional culture medium, a mixture of culture medium, adhesive cells and beads is formed. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

In the second embodiment, a part of the culture solution C is so that the lower part of the bead deposit L is immersed in the part of the culture solution C (that is, the upper part of the bead deposit L is exposed to the culture space S). ), Then the rest of the culture solution C is supplied so that the entire bead deposit L is immersed in the culture solution C (that is, the upper part of the bead deposit L is not exposed to the culture space S). Is preferable. The fixed time for the adhesive cells to adhere to the bead deposit is preferably 60 minutes or more and 2880 minutes or less, more preferably 120 minutes or more and 1440 minutes or less, and even more preferably 360 minutes or more and 1440 minutes or less.

In the third embodiment, the second material M2 is bead powder, and the second material M2 is supplied after the supply of the first material M1, or the supply of the first material M1 and the supply of the second material M2. Supply is done at the same time. The amount of second material supplied is adjusted to form the bead deposit L. The amount of the first material M1 supplied is adjusted so that the entire bead deposit L is immersed in the culture medium contained in the first material M1. The supply of the first material M1 and the supply of the second material form a mixture of culture medium, adhesive cells and beads. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

In the fourth embodiment, the second material M2 is a bead dispersion, and the first material M1 is supplied after the supply of the second material M2, or the second material M2 is supplied after the supply of the first material M1. Is supplied, or the supply of the first material M1 and the supply of the second material M2 are performed at the same time. The amount of second material supplied is adjusted to form the bead deposit L. The amount of the first material M1 supplied is adjusted so that the entire bead deposit L is immersed in the culture medium contained in the first material M1. The supply of the first material M1 and the supply of the second material form a mixture of culture medium, adhesive cells and beads. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

Hereinafter, a case where the step (a) is carried out using the cell culture apparatus 1B will be described.

When the step (a) is carried out using the cell culture apparatus 1B, the first material supply unit 22 supplies the first material M1 containing the culture solution and the adhesive cells to the culture space S of the culture tank 21. An additional culture solution may be supplied to the culture space S of the culture tank 21 by the culture solution supply unit 24b.

When the additional culture solution is supplied by the culture solution supply unit 24b, the supply of the additional culture solution may be performed before the supply of the first material M1 or after the supply of the first material M1. It may be carried out at the same time as the supply of the first material M1, but it is usually carried out after the supply of the first material M1.

In the fifth embodiment, no additional culture solution is supplied after the supply of the first material M1. The amount of the first material M1 supplied is adjusted so that the entire bead deposit L is immersed in the culture medium contained in the first material M1. It is preferable that the bead deposit L is maintained during the supply of the first material M1, but it is not essential that the bead deposit L is maintained during the supply of the first material M1.

In the fifth embodiment, when the bead deposit L is maintained during the supply of the first material M1, the supply of the first material M1 contains the adhesive cells, the culture medium C, and the bead deposit L. The culture tank 21 is prepared.

In a fifth embodiment, if the bead deposit L is not maintained during the supply of the first material M1, a mixture of culture medium, adhesive cells and beads is formed. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

In the sixth embodiment, after the supply of the first material M1, the additional culture solution is supplied. The amount of the first material M1 supplied is such that the lower part of the bead deposit L is immersed in the culture solution contained in the first material M1 (that is, the upper part of the bead deposit L is above the liquid level of the culture solution). Adjusted (to be exposed to space). The amount of the additional culture solution supplied is adjusted so that the entire bead deposit L is immersed in the culture solution (the culture solution contained in the first material M1 and the additional culture solution). That is, a part of the culture solution C is exposed so that the lower part of the bead deposit L is immersed in the part of the culture solution C (that is, the upper part of the bead deposit L is exposed in the space above the liquid surface of the culture solution C. The rest of the culture solution C is then supplied so that the entire bead deposit L is immersed in the culture solution C (ie, the upper part of the bead deposit L is not exposed to the culture space S). Be supplied. The entire bead deposit L may be finally immersed in the culture solution C, and the amount of the first material M1 supplied and the amount of the additional culture solution supplied can be adjusted as appropriate. It is preferable that the bead deposit L is maintained during the supply of the first material M1 and the supply of the additional culture medium, but the bead deposit L is maintained during the supply of the first material M1 and the supply of the additional culture solution. It is not essential to be maintained.

In the sixth embodiment, when the bead deposit L is maintained during the supply of the first material M1 and the additional culture medium, the adhesive cells and the adhesive cells are supplied by the supply of the first material M1 and the additional culture medium. A culture tank 21 containing the culture solution C and the bead deposit L is prepared.

In the sixth embodiment, if the bead deposit L is not maintained during the supply of the first material M1 and the additional culture medium, a mixture of culture medium, adhesive cells and beads is formed. In this mixture, the adhesive cells and beads are dispersed in the culture medium. After the mixture of the culture solution, the adhesive cells and the beads is formed, the cells are settled in the culture solution by gravity and settled on the bottom wall portion 211 of the culture tank 21 by allowing the culture tank 21 to stand. As a result, the bead deposit L is formed. In this way, the culture tank 21 containing the adhesive cells, the culture solution C, and the bead deposit L is prepared.

In the sixth embodiment, a part of the culture solution C is so that the lower part of the bead deposit L is immersed in the part of the culture solution C (that is, the upper part of the bead deposit L is above the liquid level of the culture solution C). The balance of the culture solution C is then supplied so that the entire bead deposit L is immersed in the culture solution C (that is, the upper part of the bead deposit L is the solution of the culture solution C). It is preferably supplied (so as not to be exposed to the space above the surface). The fixed time for the adhesive cells to adhere to the bead deposit is preferably 60 minutes or more and 2880 minutes or less, more preferably 120 minutes or more and 1440 minutes or less, and even more preferably 360 minutes or more and 1440 minutes or less.

In the present embodiment, the supply of the first material M1, the supply of the second material M2, and the supply of the additional culture solution are performed by the first material supply unit 22, the second material supply unit 23, and the culture solution supply unit 24b, respectively. The supply of the first material M1, the supply of the second material M2, and the supply of the additional culture medium may be performed manually. Further, in the present embodiment, the culture solution and the adhesive cells are supplied in the form of a mixture (that is, the first material M1), but the culture solution and the adhesive cells may be supplied separately. In the embodiment in which the culture solution and the adhesive cells are separately supplied, the adhesive cells may be supplied after the culture solution is supplied, or the culture solution supply unit may be supplied after the adhesive cells are supplied. ..

The step (b) is a step of statically culturing the adherent cells in the culture tank 21 in a state of being adhered to the bead deposit L. The step (b) is carried out in the same manner regardless of whether the cell culture device 1A is used or the cell culture device 1B is used.

The bead deposit L formed in the step (a) is also maintained in the step (b), and in the step (b), the adhesive cells are statically adhered to the bead deposit L in the culture tank 21. Incubate. In order to maintain the bead deposit L, the culture solution is not agitated in the step (b), but the culture solution supply / discharge treatment is performed as described later.

In the step (a), the culture tank 21 is allowed to stand in order to form the bead deposit L, but in the step (b), the culture tank 21 is still allowed to stand. As a result, the adhesive cells adhere to the bead deposit L and proliferate. "Adhesive cells adhere to the bead deposit L" means that the adhesive cells adhere to the surface of the beads constituting the bead deposit L. In the bead deposit L, unlike the case where the culture solution is agitated and the beads are suspended in the culture solution, one bead and another bead are present adjacent to each other, and the surface of one bead and another bead are present. It is continuous with the surface. Therefore, when the adhesive cells proliferate, the adhesive cells can proliferate while moving from the surface of one bead to the surface of another bead adjacent to the bead. Bead diameter from the perspective of allowing adherent cells to migrate from the surface of one bead to the surface of another bead adjacent to the bead within a few days (eg, about 3 days). Is preferably 5.0 mm or less, more preferably 4.0 mm or less, and even more preferably 3.0 mm or less.

The culture time in the step (b) can be appropriately adjusted according to the type of adhesive cells and the like, the total surface area of the bead deposit L and the like. The culturing time is preferably 48 hours or more and 240 hours or less, and more preferably 72 hours or more and 96 hours or less.

In step (b), during the static culture, the culture solution supply process and / or the culture solution discharge process is performed on the culture tank 21 while maintaining the bead deposit L.

In one embodiment, the culture tub 21 is first subjected to a culture broth discharge treatment, and then the culture tub 21 is subjected to a culture broth supply treatment. The cycle of the culture solution discharge treatment and the culture solution supply treatment may be repeated twice or more.

In the culture solution discharge process, the culture solution existing in the culture space S of the culture tank 21 is discharged. In the present embodiment, the culture solution discharge treatment is performed by the culture solution discharge unit 24a, but it may be performed manually. The culture solution discharged from the culture tank 21 is usually a culture solution (used culture solution) after being used for culturing adhesive cells. The discharged culture solution may be the culture solution contained in the first material M1 supplied by the culture material supply unit 22, or may be the culture solution supplied by the culture solution supply unit 24b. It may be a mixture of these.

The culture broth discharge treatment is performed under conditions in which the bead deposit L is maintained. The bead deposit L can be maintained by adjusting the rate of descent of the liquid level in the culture liquid discharge treatment. From the viewpoint of maintaining the bead deposit L, the rate of descent of the liquid level in the culture solution discharge treatment is preferably 30 mm / min or less, more preferably 10 mm / min or less, and even more preferably 1 mm / min or less.

In the culture solution supply process, the culture solution is supplied to the culture space S of the culture tank 21. In the present embodiment, the culture solution supply process is performed by the culture solution supply unit 24b, but may be performed manually. The culture solution supplied to the culture tank 21 is usually a culture solution (fresh culture solution) that is not used for culturing adhesive cells. The culture solution supplied to the culture tank 21 is supplied to the culture solution (for example, the first material M1 supplied by the first material supply unit 22) after being used for culturing adhesive cells in the culture space S of the culture tank 21. It may be a contained culture solution). That is, the culture solution may be temporarily collected from the culture space S of the culture tank 21 and supplied again to the culture space S of the culture tank 21. By temporarily collecting the culture solution from the culture space S of the culture tank 21 and supplying it to the culture space S of the culture tank 21 again, the culture solution in the culture tank 21 can be agitated. In the present embodiment, the culture solution supply unit 24b temporarily collects the culture solution from the culture space S of the culture tank 21 and resupplys the culture solution to the culture space S of the culture tank 21. That is, the culture solution supply unit 24b temporarily collects a part of the culture solution from the culture tank 21 and stores it in the tank 241b, and then supplies it to the culture space S of the culture tank 21 again.

The culture solution feeding process is performed under conditions in which the bead deposit L is maintained. The bead deposit L can be maintained by adjusting the rate of rise of the liquid level in the culture liquid supply treatment. From the viewpoint of maintaining the bead deposit L, the rate of rise of the liquid level in the culture solution supply treatment is preferably 30 mm / min or less, more preferably 10 mm / min or less, and even more preferably 1 mm / min or less.

In order to maintain the state in which the bead deposit L is formed, the culture solution discharge treatment and / or the culture solution supply treatment is performed through the opening 214 formed in the bottom wall portion 211 of the culture tank 21. The bead deposit L existing in the culture space S of the culture tank 21 is maintained by performing the culture liquid supply treatment and / or the culture liquid discharge treatment through the opening 214 formed in the bottom wall portion 211 of the culture tank 21. Easy to be done. During the culture solution discharge process and the culture solution supply process, the leakage of beads from the culture tank 21 is prevented by the filter F provided in the opening 214.

The cell culture method according to the present embodiment is a step of measuring the lactic acid concentration in the culture solution existing in the culture tank 21 and determining the continuation or termination of the static culture of the adhesive cells based on the measured lactic acid concentration. It is preferable to further contain. The measurement of lactate concentration may be performed at any time during the period during which the adhesive cells are cultured. The lactic acid concentration can be measured, for example, in step (b). The lactic acid concentration can be measured by the lactic acid concentration measuring unit 25. For example, the culture solution existing in the culture space S of the culture tank 21 by the lactic acid concentration measuring unit 25 (for example, the culture solution supplied by the culture material supply unit 22, the culture solution supplied by the culture solution supply unit 24b, or A part of (a mixture of these) is collected, and the lactic acid concentration in a part of the collected culture medium is measured. Part of the culture broth is preferably taken from the culture broth present above the bead deposits. It is preferable to determine the continuation or termination of the static culture of the adhesive cells in the culture tank 21 based on the lactate concentration measured by the lactate concentration measuring unit 25. Such a determination can be made by the control unit 3. Lactic acid concentration is an index of cell mass. That is, the higher the lactic acid concentration, the larger the cell mass. Therefore, for example, when the lactic acid concentration is equal to or higher than the predetermined reference value, the static culture of the adhesive cells is terminated, and when the lactic acid concentration is less than the predetermined reference value, the static culture of the adhesive cells is terminated. To continue.

After the static culture of the adhesive cells is completed, a cell detachment step of detaching the adhesive cells from the beads and a cell recovery step of collecting the adhesive cells detached from the beads may be performed.

In the cell detachment step, the cell detachment solution P is supplied to the culture space S of the culture tank 21 by the cell detachment solution supply unit 26 to detach the adhesive cells from the beads. In order to improve the peeling efficiency, the culture solution existing in the culture space S of the culture tank 21 may be stirred. The agitation of the culture solution can be performed, for example, by temporarily collecting the culture solution from the culture space S of the culture tank 21 and supplying the culture solution to the culture space S of the culture tank 21 again. In the present embodiment, the culture solution supply unit 24b temporarily collects the culture solution from the culture space S of the culture tank 21 and resupplys the culture solution to the culture space S of the culture tank 21. That is, the culture solution supply unit 24b temporarily collects a part of the culture solution from the culture tank 21 and stores it in the tank 241b, and then supplies it to the culture space S of the culture tank 21 again. The cell detachment step detaches the adherent cells from the beads to form a mixture of culture medium, adherent cells and beads. In the formed mixture, the adherent cells are dispersed in the culture medium together with the beads.

In the cell recovery step, the culture solution existing in the culture space S of the culture tank 21 is discharged from the culture space S of the culture tank 21 together with the adhesive cells dispersed in the culture solution by the culture solution discharge unit 24a, and is collected. Will be done. Leakage of beads from the culture tank 21 during discharge of the culture solution is prevented by a filter F provided in the opening 214.

[Reference Example 1]
HADSC-human adipose-derived stem cells (Lonza Japan Co., Ltd., Catalog No. PT-5006), which are mesenchymal stem cells, were used as adhesive cells.

A culture medium suitable for culturing adhesive cells was prepared and used. The culture solution before being used for culturing does not contain lactic acid.

A 12-well plate (manufactured by eppendolf, catalog number 0030721110) used for normal flat culture was used as a culture vessel.

Adhesive cells were seeded in the culture vessel so that the density of the adhesive cells was 1000 cells / cm ^2. After seeding, the culture vessel was allowed to stand in an incubator _{, and the adhesive cells were cultured under the conditions of 5% CO 2} concentration, temperature 37 ° C., and humidity 95% or higher. After the start of culturing, the number of cells was counted and the lactic acid concentration was measured from the 1st day to the 7th day. The number of cells was collected by peeling the cells from the culture vessel, and the number of cells contained in the cell suspension was counted by a cell counter. The lactic acid concentration was measured by collecting the culture broth from the culture vessel and using a high performance liquid chromatograph mass spectrometer LCMS-8050 (Shimadzu Corporation).

When the density of adhesive cells is 3000 cells / cm ² and the density of adhesive cells is 6000 cells / cm ² , the same as when the density of adhesive cells is 1000 cells / cm ^2. , Adhesive cells were cultured, the number of cells was measured, and the lactic acid concentration was measured.

The measurement results are shown in FIG. In FIG. 5, the lactic acid concentration is represented by an internal standard method using 2-isopropylmalic acid as an internal standard based on LC / MS / MS method package cell culture profiling by Shimadzu Corporation. The internal standard method is a quantitative method for determining the concentration of a target component based on the relationship between the peak area ratio and the concentration ratio of the target component and the internal standard substance.

As shown in FIG. 5, there is a correlation between the number of cells and the lactate concentration. Lactic acid contained in the culture medium is a cell metabolite secreted from cells. Therefore, the number of cells can be estimated by measuring the concentration of lactic acid contained in the culture medium.

[Test Example 1]
HADSC-human adipose-derived stem cells (Lonza Japan Co., Ltd., Catalog No. PT-5006), which are mesenchymal stem cells, were used as adhesive cells.

A culture medium suitable for culturing adhesive cells was prepared and used. The culture solution before being used for culturing does not contain lactic acid.

Beads with a diameter of 3 mm were used as a carrier for culturing adhesive cells. As the beads having a diameter of 3 mm, spherical polystyrene beads prepared by hydrophilizing the surface with oxygen plasma after injection molding were used. The beads are observed using a phase-contrast microscope, the area of the beads taken from directly above is quantified, and assuming that the beads are true spheres, the diameter of the beads is calculated from the quantified area value. Was calculated. The specific gravity of polystyrene beads is about 1.04.

A 10 mL pipette (Corning International Co., Ltd., Catalog No. 357551) was used as a culture tank. A 10 mL pipette was used by connecting a 10 mL disposable syringe (Terumo Corporation, Catalog No. SS-10ESZ) to the opening at the tip thereof via a silicone tube.

After accommodating 10 mL of the culture solution, 2.1 × 10 ⁵ adhesive cells, and 176 beads having a diameter of 3 mm in the culture tank, the cells were allowed to stand to form bead deposits. The culture solution and adhesive cells were introduced into the culture tank via a syringe and a tube. That is, the syringe containing the culture solution and the adhesive cells was connected to the 10 mL pipette via the silicone tube, and the culture solution and the adhesive cells were introduced into the culture tank. The height of the culture solution was 319 mm, the thickness of the bead deposit was 130 mm, and the number of layers of the bead deposit was 43.3 layers. The number of layers of the bead deposit was calculated by dividing the thickness of the bead deposit (130 mm) by the diameter of the beads (3 mm).

Adhesive cells were cultured while the culture tank was allowed to stand as it was to maintain the state in which bead deposits were formed. During the culture, the culture solution was not stirred. Adhesive cells were cultured for 96 hours.

After culturing the adhesive cells, the culture solution was aspirated and collected from the lower layer side of the bead deposit, 1 mL each, 8 times in total with a syringe connected to the tip opening of the pipette. The culture broth collected 8 times in total, 1 mL each from the lower layer side of the bead deposit, is referred to as the culture broth of the 1st to 8th regions, respectively. The culture solution in the first region is located on the lowermost layer side, and the culture solution in the eighth region is located on the uppermost layer side. The lactic acid concentration in the culture broths in the 1st to 8th regions was measured using a high performance liquid chromatograph mass spectrometer LCMS-8050 (Shimadzu Corporation). The measurement results are shown in Table 1. In Table 1, the lactic acid concentration is represented by an internal standard method using 2-isopropylmalic acid as an internal standard based on LC / MS / MS method package cell culture profiling by Shimadzu Corporation. The internal standard method is a quantitative method for determining the concentration of a target component based on the relationship between the peak area ratio and the concentration ratio of the target component and the internal standard substance.

As shown in Table 1, it was observed that the lactic acid concentration tended to increase toward the lower layer side of the bead deposit. From the above results, it can be seen that lactic acid, which is a waste product produced by the cells, stays around the cells without diffusing toward the lower layer side of the bead deposit.

[Test Example 2]
HADSC-human adipose-derived stem cells (Lonza Japan Co., Ltd., Catalog No. PT-5006), which are mesenchymal stem cells, were used as adhesive cells.

A culture medium suitable for culturing adhesive cells was prepared and used. The culture solution before being used for culturing does not contain lactic acid.

Beads with a diameter of 0.250 mm were used as a carrier for culturing adhesive cells. Soluble microcarriers coated with denatured collagen (Corning International, Inc., Catalog No. 4979) were used as beads with a diameter of 0.250 mm. The beads are observed using a phase-contrast microscope, the area of the beads taken from directly above is quantified, and assuming that the beads are true spheres, the diameter of the beads is calculated from the quantified area value. Was calculated.

As culture tanks, a 12-well plate (manufactured by eppendolf, catalog number 0030721110), a 24-well plate (manufactured by eppendolf, catalog number 0030722019), a 48-well plate (manufactured by eppendolf, catalog number 0030723112) and a 96-well plate (manufactured by eppendolf). , Catalog number 0030502345) was used.

Each culture vessel, the culture solution 1 mL, ⁴ adherent cells 6.7 × 10, after receiving the ^four beads 3.4 × 10 diameter 0.250 mm, to form a bead deposit on standing.

When a 12-well plate was used as the culture tank, the height of the culture solution was 2.6 mm and the thickness of the bead deposit was 1.1 mm. The number of bead deposits was 4.4. The number of layers of bead deposits was calculated by dividing the thickness of the bead deposits (1.1 mm) by the diameter of the beads (0.250 mm).

When a 24-well plate was used as the culture tank, the height of the culture solution was 4.8 mm and the thickness of the bead deposit was 2.1 mm. The number of bead deposits was 8.3. The number of layers of bead deposits was calculated by dividing the thickness of the bead deposits (2.1 mm) by the diameter of the beads (0.250 mm).

When a 48-well plate was used as the culture tank, the height of the culture solution was 11.7 mm and the thickness of the bead deposit was 5.1 mm. The number of bead deposits was 20.3. The number of layers of bead deposits was calculated by dividing the thickness of the bead deposits (5.1 mm) by the diameter of the beads (0.250 mm).

When a 96-well plate was used as the culture tank, the height of the culture solution was 35.4 mm and the thickness of the bead deposit was 15.4 mm. The number of bead deposits was 61.4. The number of layers of bead deposits was calculated by dividing the thickness of the bead deposits (15.4 mm) by the diameter of the beads (0.250 mm).

Adhesive cells were cultured while the culture tank was allowed to stand as it was to maintain the state in which bead deposits were formed. During the culture, the culture solution was not stirred. Adhesive cells were cultured for 8 days.

After culturing the adherent cells, a culture solution (a part of the culture solution existing above the bead deposit) is collected from each culture tank over time, and the lactic acid concentration in each culture solution is measured by a high-performance liquid chromatograph mass spectrometer LCMS. It was measured using -8050 (Shimadzu Seisakusho Co., Ltd.). The measurement results are shown in Table 2. In Table 2, the lactic acid concentration is represented by an internal standard method using 2-isopropylmalic acid as an internal standard based on LC / MS / MS method package cell culture profiling by Shimadzu Corporation. The internal standard method is a quantitative method for determining the concentration of a target component based on the relationship between the peak area ratio and the concentration ratio of the target component and the internal standard substance. Table 2 also shows the measurement results when a 12-well plate was used as the culture tank and the adhesive cells were cultured without using beads having a diameter of 0.250 mm (hereinafter referred to as "planar culture"). ..

As shown in Table 2, it was observed that the lactic acid concentration tended to decrease as the number of layers of the bead deposits increased. From the above results, it can be seen that as the number of layers of bead deposits increases, the accumulation of waste products lowers the cell proliferation performance.

[Examples 1 to 4 and Comparative Example 1]
HADSC-human adipose-derived stem cells (Lonza Japan Co., Ltd., Catalog No. PT-5006), which are mesenchymal stem cells, were used as adhesive cells.

Cellartis ™ MSC Xeno-Free Culture Medium (manufactured by Takara Bio Inc., Catalog No. Y50200) was used as a culture medium for culturing adherent cells.

Diameter 0.250 mm (Comparative Example 1), Diameter 0.789 mm (Example 1), Diameter 1.588 mm (Example 2), Diameter 3.175 mm (Example 3) and Diameter 4.763 mm (Example 4). Five types of beads were used as carriers for culturing adhesive cells. For beads with a diameter of 0.250 mm, soluble microcarriers coated with denatured collagen (Corning International, Inc., Catalog No. 4979) were used. Regarding the other four types of beads, the surface of acrylic beads (Humanity Co., Ltd.) was coated with Vitronectin-N (Thermo Fisher SCIENTIFIC, Catalog No. A14700) and used. The coating of vitronectin-N was carried out according to the protocol (after diluting 100 times with DPBS, the acrylic beads were immersed, allowed to stand at room temperature for 1 hour, and then used without washing and drying). The beads are observed using a phase-contrast microscope, the area of the beads taken from directly above is quantified, and assuming that the beads are true spheres, the diameter of the beads is calculated from the quantified area value. Was calculated.

The tip opening of a 10 mL pipette (Corning International Co., Ltd., Catalog No. 357551) was closed by heat and used as a culture tank.

After stacking one or two layers of beads in a container having a wide flat surface, adhesive cells suspended in a culture medium were introduced. After introduction, the container was _{allowed to stand overnight in an incubator having a 5% CO 2} concentration, a temperature of 37 ° C., and a humidity of 95% or more, and adhesive cells were initially adhered to the beads. At this time, for each bead, the amount of beads and the number of cells were adjusted so that the number of introduced cells per unit surface area of the beads was the same.

On the first day, after introducing the culture solution and the initially adhered beads into the culture tank, the beads were allowed to stand to form bead deposits.

The amount of the culture solution introduced is such that the thickness of the culture solution is 2 mm (that is, the introduction of the culture solution) assuming that the culture is flat-cultured with the culture area (total surface area of the beads) calculated from the diameter and number of the beads to be introduced. The amount (μL) = culture area (mm ² ) × 2 mm) was adjusted. The bead deposits are reliably immersed in the culture medium.

The amount of beads introduced with a diameter of 0.250 mm was adjusted so that the number of bead deposits was 2.6, 7.8, 15.6, or 23.5. The number of layers of the bead deposit is calculated by dividing the thickness of the bead deposit by the diameter of the beads (0.250 mm).

The amount of beads introduced with a diameter of 0.789 mm was adjusted so that the number of bead deposits was 2.6, 7.8, 15.6, or 23.5. The number of layers of the bead deposit is calculated by dividing the thickness of the bead deposit by the diameter of the beads (0.789 mm).

The amount of beads introduced with a diameter of 1.588 mm was adjusted so that the number of bead deposits was 2.6, 7.8, 15.6, or 23.5. The number of layers of the bead deposit is calculated by dividing the thickness of the bead deposit by the diameter of the beads (1.588 mm).

The amount of beads introduced with a diameter of 3.175 mm was adjusted so that the number of bead deposits was 2.6 or 7.8. The number of layers of the bead deposit is calculated by dividing the thickness of the bead deposit by the diameter of the beads (3.175 mm).

The amount of beads introduced with a diameter of 4.763 mm was adjusted so that the number of bead deposits was 2.6 or 7.8. The number of layers of the bead deposit is calculated by dividing the thickness of the bead deposit by the diameter of the beads (4.763 mm).

After the formation of the bead deposits, the culture tank _{was allowed to stand in an incubator having a 5% CO 2} concentration, a temperature of 37 ° C., and a humidity of 95% or more to start culturing. Gender) was compared.

In order to compare cell proliferation (cell productivity), a part of the culture solution (a part of the culture solution existing above the bead deposit) was collected from each culture tank, and the lactic acid concentration was measured by LCMS-. This was performed using 8050 (Shimadzu Corporation). For each bead, the lactic acid concentration when the number of layers of the bead deposit was 2.6 was used as a reference, and the lactic acid concentration was standardized when the number of layers was other than that.

The measurement results are shown in Table 3. In Table 3, the lactic acid concentration is represented by an internal standard method using 2-isopropylmalic acid as an internal standard based on LC / MS / MS method package cell culture profiling by Shimadzu Corporation. The internal standard method is a quantitative method for determining the concentration of a target component based on the relationship between the peak area ratio and the concentration ratio of the target component and the internal standard substance.

When beads with a diameter of 0.250 mm are used (Comparative Example 1), when the number of layers of the bead deposit exceeds 2.6, the lactic acid concentration when the number of layers of the bead deposit is 2.6 is used as a reference. As a result, it was not possible to achieve a lactate concentration of 80% or more (that is, the desired cell proliferation could not be achieved). When beads with a diameter of 0.250 mm are used (Comparative Example 1), if the number of beads deposits exceeds 2.6, the effects of nutrient depletion, waste retention, etc. that occur in the bead deposits are large. It is probable that a lactic acid concentration of 80% or more could not be achieved based on the lactic acid concentration when the number of bead deposits was 2.6. On the other hand, when beads having a diameter of 0.789 mm, 1.588 mm, 3.175 mm and 4.763 mm are used (Examples 1 to 4), even if the number of beads deposits exceeds 2.6 layers. The lactic acid concentration of 80% or more could be achieved based on the lactic acid concentration when the number of layers of the bead deposit was 2.6 (that is, the desired cell proliferation was achieved). ). When beads having a diameter of 0.789 mm, 1.588 mm, 3.175 mm and 4.763 mm are used (Examples 1 to 4), the effects of nutrient depletion, waste retention, etc. that occur in the bead deposits are small, so the beads It is considered that 80% or more of the lactic acid concentration could be achieved based on the lactic acid concentration when the number of layers of the sediment was 2.6.

Claims (16)

A cell culture device including a culture unit for statically culturing adhesive cells and a control unit for controlling the operation of the culture unit.
The culture section
Incubator and
A first material supply unit that supplies the first material to the culture tank,
A second material supply unit that supplies the second material to the culture tank,
A culture solution supply / discharge unit that performs a culture solution supply process and / or a culture solution discharge process to the culture tank.
With
The first material contains a culture medium and adhesive cells.
The second material comprises beads having a diameter of 0.7 mm or more having a surface to which the adhesive cells can adhere.
An opening is formed in the bottom wall of the culture tank.
The culture solution supply / discharge unit performs the culture solution supply process and / or the culture solution discharge process through the opening.
In the control unit, the first material and the second material are supplied to the culture tank by the first material supply unit and the second material supply unit, and then, in the culture tank, the adhesive cells are subjected to. The culture solution was statically cultured in a state of being adhered to the bead deposit containing beads having a diameter of 0.7 mm or more, and the culture solution was supplied by the culture solution supply / discharge unit while the bead deposit was maintained during the static culture. The cell culture apparatus that controls the operation of the culture unit so that the treatment and / or the culture solution discharge treatment is performed. A cell culture device including a culture unit for statically culturing adhesive cells and a control unit for controlling the operation of the culture unit.
The culture section
Incubator and
A first material supply unit that supplies the first material to the culture tank,
The bead deposits housed in the culture tank and
A culture solution supply / discharge unit that performs a culture solution supply process and / or a culture solution discharge process to the culture tank.
With
The first material contains a culture medium and adhesive cells.
The bead deposit comprises beads having a diameter of 0.7 mm or more having a surface to which the adhesive cells can adhere.
An opening is formed in the bottom wall of the culture tank.
The culture solution supply / discharge unit performs the culture solution supply process and / or the culture solution discharge process through the opening.
In the control unit, the first material is supplied to the culture tank by the first material supply unit, and then the adherent cells are statically cultured in the culture tank in a state of being adhered to the bead deposit. Then, during the static culture, the operation of the culture unit is performed so that the culture solution supply process and / or the culture solution discharge process is performed by the culture solution supply / discharge unit while the bead deposit is maintained. The cell culture apparatus to be controlled. The cell culture apparatus according to claim 1 or 2, wherein the thickness of the bead deposit is three times or more the diameter of the beads. The cell culture apparatus according to any one of claims 1 to 3, wherein the beads have a diameter of 5 mm or less. The control unit controls the operation of the culture solution supply / discharge unit so that the rate of increase in the liquid level in the culture solution supply process and / or the rate of decrease in the liquid level in the culture solution discharge process is 30 mm / min or less. The cell culture apparatus according to any one of claims 1 to 4. The cell culture apparatus according to any one of claims 1 to 5, wherein the peripheral wall portion of the culture tank is inclined toward the opening of the bottom wall portion. The cell culture apparatus according to any one of claims 1 to 6, wherein a filter for preventing leakage of the beads from the culture tank is provided in the opening. The control unit is supplied with the first material in an amount that exposes the upper part of the bead deposit by the first material supply unit, and then a certain period of time elapses for the adhesive cells to adhere to the bead deposit. Later, while the bead deposits were maintained, the culture broth was supplied in an amount such that the upper part of the bead deposits was not exposed by the culture broth supply / discharge unit, and then the adhesive cells were transferred in the culture tank. The cell culture apparatus according to any one of claims 1 to 7, which controls the operation of the culture unit so that the cell culture unit is statically cultured in a state of being adhered to the bead deposit. The culture unit further includes a lactic acid concentration measuring unit for measuring the lactic acid concentration in the culture solution existing in the culture tank.
The cell according to any one of claims 1 to 8, wherein the control unit determines the continuation or termination of the static culture of the adhesive cell based on the lactate concentration measured by the lactate concentration measuring unit. Culture device. The following process:
(A) A step of preparing a culture tank containing an adhesive cell, a culture solution, and a bead deposit; and (b) a state in which the adhesive cell is adhered to the bead deposit in the culture tank. It is a cell culture method including a step of statically culturing in.
The bead deposit comprises beads having a diameter of 0.7 mm or more having a surface to which the adhesive cells can adhere.
An opening is formed in the bottom wall of the culture tank.
The cell culture method in which a culture solution supply process and / or a culture solution discharge process is performed on the culture tank through the opening while the bead deposit is maintained during the static culture. The cell culture method according to claim 10, wherein the thickness of the bead deposit is three times or more the diameter of the beads. The cell culture method according to claim 10 or 11, wherein the beads have a diameter of 5 mm or less. The cell culture method according to any one of claims 10 to 12, wherein the rate of increase in the liquid level in the culture solution supply process and / or the rate of decrease in the liquid level in the culture solution discharge process is 30 mm / min or less. The cell culture method according to any one of claims 10 to 13, wherein a filter for preventing leakage of the beads from the culture tank is provided at the opening. In step (a), a part of the culture solution is supplied in an amount that exposes the upper part of the bead deposit, and then after a certain period of time for the adhesive cells to adhere to the bead deposit, the bead The culture containing the adherent cells, the culture solution, and the bead deposit by supplying the rest of the culture solution in an amount that does not expose the upper part of the bead deposit while maintaining the deposit. The cell culture method according to any one of claims 10 to 14, wherein a tank is prepared. 10.15 of claims 10 to 15, further comprising a step of measuring the lactic acid concentration in the culture solution existing in the culture tank and determining the continuation or termination of the static culture of the adhesive cells based on the measured lactic acid concentration. The cell culture method according to any one of the above.

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