WO2017212829A1

WO2017212829A1 - Cell culturing method and liquid culture medium

Info

Publication number: WO2017212829A1
Application number: PCT/JP2017/017040
Authority: WO
Inventors: 橋本　斉和; 晴貴冨川
Original assignee: 富士フイルム株式会社
Priority date: 2016-06-06
Filing date: 2017-04-28
Publication date: 2017-12-14
Also published as: JPWO2017212829A1; JP6758371B2

Abstract

Provided are: a cell culturing method comprising a heating step in which a liquid culture medium having a heating phase transition temperature Tc and a cooling phase transition temperature Tcs that is lower than Tc is heated from less than Tc to at least Tc, and a culturing step in which, after the liquid culture medium has been heated to at least Tc in the heating step, cells are suspension-cultured in the liquid culture medium at a culturing temperature of at least Tcs; and a liquid culture medium having a heating phase transition temperature Tc and a cooling phase transition temperature Tcs.

Description

Cell culture method and culture solution

The present invention relates to a cell culture method and a culture solution.

As a scaffold material for culturing cells, it is known to use nanofibers such as cellulose nanofibers.
For example, in Patent Document 1, mesenchymal stem cells are dispersed in a cell culture solution in which a polysaccharide derived from a natural product such as cellulose nanofiber is dispersed so as to float in the solution, thereby freezing the culture solution. Without inhibiting the differentiation of mesenchymal stem cells under culture conditions, and can maintain and preserve the initial state for a desired period of time, and polysaccharides in the form of nanofibers such as cellulose nanofibers It is described that mesenchymal stem cells can be collected from a culture solution by using a natural product-derived polysaccharide, and can be reused. ([0011]).
Patent Document 2 also describes a cell culture or cell delivery composition comprising plant-derived mechanically disrupted cellulose nanofibers and / or derivatives thereof in the form of hydrogels or membranes, further providing cells A method of culturing cells comprising the steps of contacting the cells with the cell culture or cell delivery composition to form a matrix, and culturing the cells in a three-dimensional or two-dimensional arrangement within the matrix, and Cellulose nanofibers are removed by providing a cell culture medium and a cell-containing material, contacting the cell culture material with a degrading enzyme, centrifuging the material to precipitate cells and cell aggregates, and decantation. From cell culture materials to plant-based cellulose nanofibers and / or How to remove the culture have been described (claims).

International Publication No. 2015/111734 Special table 2013-54156 gazette

In the conventional method for recovering cultured cells, suspended cells were subjected to centrifugation, and the precipitated cells were recovered (Patent Documents 1 and 2).
However, since the cells are crushed and damaged by the centrifugal acceleration applied to the cells during the centrifuge treatment, it has been a problem that the cell growth rate and the cell recovery rate cannot be compatible.
In addition, there is a disadvantage that the culture cost increases due to an increase in the number of steps that require complicated operations, such as the need to dilute the cell dispersion obtained by culturing in order to perform centrifugation. .

Therefore, the present invention suppresses damage to cultured cells during recovery, can achieve both cell growth rate and cell recovery rate, and can simplify complicated recovery operations. It is another object of the present invention to provide a culture solution used for this culture method.

As a result of intensive studies to solve the above problems, the present inventors have used a culture solution that undergoes a phase transition from sol to gel at a high temperature and a phase transition from gel to sol at a low temperature. When cells are suspended in culture at a temperature higher than the phase transition from gel to sol after the phase transition to, the cultured cells are allowed to settle simply by lowering the culture solution below the temperature at which the phase transition from gel to sol occurs. It is possible to suppress the damage that cultured cells receive during recovery, to achieve both cell growth rate and cell recovery rate, and to simplify complicated recovery operations. Completed the invention.

That is, the present invention provides the following [1] to [23].
[1] A temperature raising step for raising the temperature of a culture solution having a temperature-falling phase transition temperature Tcs that is lower than the temperature-raising phase transition temperature Tc to a temperature higher than Tc from less than Tc;
In the temperature raising step, after the temperature of the culture solution is raised to Tc or higher, a culture step of suspending cells in the culture solution at a culture temperature of Tcs or higher;
A method for culturing cells.
Here, the temperature transition phase transition temperature Tc is the temperature of the culture solution when the culture solution is heated from 3.0 ° C. to 98.0 ° C. at a temperature increase rate of 5.0 ° C./min. This is the temperature at which the viscosity of the culture broth becomes 10 × ηs when the viscosity at 3.0 ° C. is ηs, and the phase transition temperature Tcs during cooling is 5.0 ° C./min for the culture broth. In the case where the temperature is lowered from 98.0 ° C. to 3.0 ° C. at the rate of temperature reduction, the temperature is the temperature at which the viscosity of the culture solution becomes 10 × ηs, the temperature unit is ° C., and the viscosity unit is Pa · s. And
[2] The method for culturing cells according to [1] above, wherein Tc and Tcs satisfy the following formula (1).
1.0 ° C ≦ Tc−Tcs ≦ 70.0 ° C (1)
[3] After the culture step,
A recovering step of lowering the temperature of the culture solution below Tcs to settle and recover the cells cultured in the culture step;
The method for culturing cells according to the above [1] or [2].
[4] The method for culturing cells according to any one of [1] to [3] above, wherein the temperature-falling phase transition temperature Tcs is 3.0 ° C. or higher and 41.0 ° C. or lower.
[5] The cell culture according to any one of [1] to [4], wherein the culture solution includes cellulose nanofibers having an average diameter of 2.0 nm to 100 nm and a thermal sol-gel changing agent. Method.
[6] The method for culturing cells according to [5] above, wherein the cellulose nanofiber has a carboxy group content of 0.60 mmol / g or more and 2.0 mmol / g or less.
[7] The method for culturing cells according to [5] or [6] above, wherein the content of the cellulose nanofiber is 0.01% by mass or more and 1.0% by mass or less in the culture solution.
[8] The cell culture method according to any one of [5] to [7], wherein the cellulose nanofibers are oxidized.
[9] The cell culture according to any one of [5] to [8], wherein the content of the thermal sol-gel change agent is 0.05% by mass or more and 3.0% by mass or less in the culture solution. Method.
[10] The cell culture method according to any one of [5] to [9] above, wherein the thermal sol-gel changing agent is methylcellulose.
[11] The method for culturing cells according to any one of [1] to [10] above, wherein the cells are stem cells.
[12] After the temperature raising step and before the culture step,
The cell according to any one of the above [1] to [11], comprising a post-gelation seeding step of seeding cells in the culture solution after raising the temperature of the culture solution to Tc or higher in the temperature raising step. Culture method.
[13] Before the temperature raising step,
The cell culturing method according to any one of [1] to [11] above, comprising a pre-gelation seeding step of seeding cells in the culture solution.
[14] A culture solution having a temperature transition phase transition temperature Tc and a temperature transition phase transition temperature Tcs.
Here, the temperature transition phase transition temperature Tc is the temperature of the culture solution when the culture solution is heated from 3.0 ° C. to 98.0 ° C. at a temperature increase rate of 5.0 ° C./min. This is the temperature at which the viscosity of the culture broth becomes 10 × ηs when the viscosity at 3.0 ° C. is ηs, and the phase transition temperature Tcs during cooling is 5.0 ° C./min for the culture broth. In the case where the temperature is lowered from 98.0 ° C. to 3.0 ° C. at the rate of temperature reduction, the temperature is the temperature at which the viscosity of the culture solution becomes 10 × ηs, the temperature unit is ° C., and the viscosity unit is Pa · s. And
[15] The culture solution according to [14], wherein Tc and Tcs satisfy the following formula (1).
1.0 ° C ≦ Tc−Tcs ≦ 70.0 ° C (1)
[16] The culture solution according to [14] or [15] above, wherein the temperature-falling phase transition temperature Tcs is 3.0 ° C. or higher and 41.0 ° C. or lower.
[17] The culture solution according to any one of [14] to [16] above, comprising cellulose nanofibers having an average diameter of 2.0 nm or more and 100 nm or less and a thermal sol-gel change agent.
[18] The culture solution according to [17], wherein the cellulose nanofiber has a carboxy group content of 0.60 mmol / g or more and 2.0 mmol / g or less.
[19] The cell culture solution according to [17] or [18], containing the cellulose nanofibers in an amount of 0.01% by mass to 1.0% by mass.
[20] The culture solution according to any one of [17] to [19], wherein the cellulose nanofibers are oxidized.
[21] The culture solution according to any one of [17] to [20] above, which contains 0.05 to 3.0% by mass of the thermal sol-gel change agent.
[22] The culture solution according to any one of [17] to [21], wherein the thermal sol-gel changing agent is methylcellulose.
[23] The culture solution according to any one of the above [14] to [22] for use in the cell culture method according to any one of the above [1] to [13].

ADVANTAGE OF THE INVENTION According to this invention, the culture | cultivation method of the cell which can suppress the damage which the cultured cell receives at the time of collection | recovery, can make a cell growth rate and a cell collection | recovery rate compatible, and can simplify complicated collection | recovery operation. In addition, a culture solution used for this culture method can be provided.

FIG. 1 is a graph showing the relationship between the temperature and viscosity of a culture solution used in the cell culture method of the present invention. When the temperature rises, the sol state transitions to the gel state at the temperature rising phase transition temperature Tc, and when the temperature falls, the gel state transitions to the sol state at the temperature falling phase transition temperature Tcs. The temperature of the culture solution is raised to T1 so that the viscosity becomes a gelled state of 10 × ηs or higher, and then the cells are cultured in suspension. After culturing, the temperature of the culture solution is lowered to T2, and the cells are sedimented in a sol state having a viscosity of 10 × ηs or more. However, T1 ≧ Tc and T2 <Tcs. The viscosity at T1 is η1, the viscosity at T2 is η2, and the viscosity at 3 ° C. when the temperature is lowered is ηs ′.

First, the advantages and advantages of the present invention over the prior art will be described.
In the conventional cell culturing method, a cultured cell dispersion obtained by suspension culture (referred to as a cell dispersion containing cultured cells and a culture solution) is subjected to a centrifugation process to precipitate the cultured cells and cultured. Cells were recovered. Therefore, in the conventional method for recovering cultured cells in which the centrifuge treatment is performed, the damage caused by the centrifugal acceleration applied to the cells during the centrifuge treatment is large, and the cell mortality rate before and after the centrifuge treatment is high.
In contrast, in the cell culture method of the present invention, the culture solution is given a function of phase transition from a sol state to a gel state at a high temperature and a phase transition from a gel state to a sol state at a low temperature. The cultured cells (including cell spheroids) can be sedimented by suspension culture in the cultured medium and sol-gelation of the culture liquid gelled at a low temperature after completion of the culture. The cells can be settled without depending on the sedimentation treatment, and the damaged cells can be prevented from being damaged during the collection, and the complicated collection operation can be simplified.

In the conventional cell culturing method, the culture solution is stirred during the culturing to prevent the cells in the culturing from sedimenting and to maintain the suspension culture.
On the other hand, in the cell culture method of the present invention, since the cells are suspended in the gelled culture solution, the suspension culture can be maintained without stirring the culture solution during the culture. In addition, cells in culture do not settle easily, and the gel of the present invention constitutes a gel that is not too strong so that it can sufficiently diffuse nutrients and oxygen necessary for growth, so that a high cell growth rate can be achieved. Can do.

In the cell culturing method of the present invention, cells (including spheroids) are suspended and cultured in a gelled culture solution, and then the temperature is lowered to a temperature below the gel-sol transition point to obtain a sol state. And facilitates sedimentation of cultured cells. Since the cultured cells can be easily settled only by the temperature operation, the temperature lowering operation is simple and the damage to the cells is small, so that a high cell sedimentation rate and a low cell mortality rate can be achieved.

In the present specification, cellulose nanofiber may be referred to as “CNF”.
In the present specification, methyl cellulose may be referred to as “MC”.
Further, in this specification, the card run (curdlan) may be referred to as “CU”.
In the present specification, carboxymethyl may be referred to as “CM”.
In this specification, 2,2,6,6-tetramethyl-1-piperidine-N-oxyl is referred to as “TEMPO”. There is a case.
In the present specification, a range represented by using “to” means a range including both ends before and after “to” in the range. For example, a and b are included in the range of “a to b” (where a and b represent a certain numerical value (real number) and a <b).
In this specification, the unit of temperature is ° C., and the unit of viscosity is Pa · s.

[Cell culture method]
Hereinafter, the cell culture method of the present invention will be described in detail.
The cell culturing method of the present invention includes a temperature raising step for raising the temperature of a culture solution from less than Tc to Tc or more, and a culture step for culturing cells in suspension in a culture solution at a culture temperature of Tcs or more.
In the cell culturing method of the present invention, after the temperature of the culture solution is raised to Tc or higher in the temperature raising step, the culture solution is kept at Tcs or higher before the cells are suspended and cultured at the culture temperature of Tcs or higher in the culturing step. It is preferable that
Moreover, it is preferable that the culture method of this invention is equipped with the collection | recovery process which cools a culture solution to less than Tcs and sediments the cultured cell.
Moreover, the culture method of the present invention may comprise a post-gelation seeding step in which cells are seeded in a culture solution after the temperature raising step, or a pre-gelation seeding step in which cells are seeded in the culture solution before the temperature raising step. preferable.
Moreover, the culture solution used for the culturing method of the present invention has a temperature transition phase transition temperature Tc and a temperature transition phase transition temperature Tcs.

<Culture solution>
The culture solution used in the cell culturing method of the present invention (hereinafter sometimes simply referred to as “the culture solution of the present invention”) will be described in detail.
The culture solution of the present invention has a temperature transition phase transition temperature Tc that is lower than the temperature transition phase transition temperature Tc and Tc.

Here, the temperature transition phase transition temperature Tc and the temperature transition phase transition temperature Tcs are defined as follows.
The temperature transition phase transition temperature Tc is the viscosity of the culture solution at 3.0 ° C. when the culture solution is heated from 3.0 ° C. to 98.0 ° C. at a temperature increase rate of 5.0 ° C./min. Is the temperature at which the viscosity of the culture solution becomes 10 × ηs.
The temperature transition phase transition temperature Tcs is a temperature at which the viscosity of the culture solution becomes 10 × ηs when the culture solution is cooled from 98.0 ° C. to 3.0 ° C. at a temperature reduction rate of 5.0 ° C./min. Temperature.
Here, Tc and Tcs satisfy the relationship of Tc> Tcs.
From the above definition, it can also be said that Tc and Tcs satisfy the relationship of 3.0 <Tc <98.0 and 3.0 <Tcs <98.0.

In the present invention, a state where the viscosity is 10 × ηs or more is referred to as “gelation state”, and a viscosity of 10 × ηs or more is referred to as “gelation”, and the viscosity is less than 10 × ηs. This state is referred to as a “solation state”, and a viscosity of less than 10 × ηs is referred to as “solation”.

The sol-gel transition of the culture solution will be described with reference to FIG.
The relationship of the temperature T-viscosity η when the culture solution is heated is represented by the lower curve of the graph of FIG. 1, and the relationship of the temperature T-viscosity η when the culture solution is lowered is the upper side of the graph of FIG. It is represented by the curve.
The viscosity at 3.0 ° C. when the temperature is raised is ηs.
First, when the temperature of the culture solution is raised and the temperature reaches Tc, the viscosity of the culture solution becomes 10 × ηs. That is, at the temperature Tc, the culture solution is in a gelled state.
When the temperature of the culture solution is raised to T1, the viscosity of the culture solution becomes η1. That is, at the temperature T1, the culture solution is in a gelled state. However, T1 is a temperature satisfying T1 ≧ Tc, and η1 is a viscosity satisfying η1 ≧ 10 × ηs.
Next, when the temperature of the culture solution is decreased from T1, and the temperature reaches Tcs, the viscosity of the culture solution becomes 10 × ηs. That is, at the temperature Tcs, the culture solution is in a gelled state.
Furthermore, when the temperature of the culture solution is lowered to T2, the viscosity of the culture solution becomes η2. That is, at the temperature T2, the culture solution is in a sol state. However, T2 is a temperature satisfying T2 <Tcs, and η2 is a viscosity satisfying η2 <10 × ηs.

The difference ΔTc = Tc−Tcs between Tc and Tcs is not particularly limited, but is preferably 1.0 ° C. ≦ ΔTc ≦ 70.0 ° C., more preferably 5.0 ° C. ≦ ΔTc ≦ 65.0 ° C. Yes, more preferably 10.0 ° C. ≦ ΔTc ≦ 60.0 ° C.
When ΔTc is within this range, the width of the sol-gel transition is wide, so that the sol-formation and the gelation hardly occur at the same time, the culture solution becomes more stable, and the cell growth rate tends to be improved.
Further, the temperature transition phase transition temperature (Tc) is unlikely to be high. Since Tc does not easily reach a high temperature, the temperature during the heat treatment of the culture solution in the temperature raising step is low, the medium components in the culture solution are hardly denatured, and the cell growth rate is likely to be improved.
Moreover, the temperature transition phase transition temperature (Tcs) is unlikely to be low. Since Tcs is unlikely to become low temperature, the temperature during the temperature lowering process in the recovery process is not lowered, and the cell mortality rate is likely to be lowered.

(Measurement method of phase transition temperature)
The viscosity of the culture solution was 3.0 using a plate viscometer (for example, MCR301 manufactured by Anton Paar) under the measurement conditions of plate diameter = 45 mm, gap = 0.049 mm, and shear rate = 0.1 s ^−1. The temperature at which the viscosity is 10 times the viscosity at 3.0 ° C. is measured as “temperature transition phase transition temperature (Tc)”. "
Next, the viscosity of the culture solution was measured from 98.0 ° C. to 3.0 ° C. under the same measurement conditions while decreasing the temperature at a temperature decrease rate of 5.0 ° C./min. The temperature which is 10 times the viscosity at is referred to as “temperature transition phase transition temperature (Tcs)”.

(Hysteresis)
In the present invention, the sol-gel transition of the culture solution is caused by heat, but the temperature transition temperature transition temperature (Tc) (sometimes referred to as “gelation temperature”) and the temperature transition temperature transition temperature (Tcs) (“sol” Is characterized by the presence of hysteresis.
As described above, since Tc and Tcs satisfy the relationship of Tc> Tcs, the culture solution of the present invention can maintain the gelled state even when the temperature is lowered, the gelation temperature is widened, and the culture temperature can be widened. It has the characteristics.
In addition, the culture solution of the present invention has a large sol-gel transition hysteresis. However, if the sol-gel transition hysteresis is large, if the culture solution is heated to a temperature higher than Tc and gelled, the sol will not exceed the Tc. It is difficult to convert and buoyancy is easy to obtain. That is, once the culture solution of the present invention is gelated by exposure to high temperature, it does not sol even if it is lowered to a low temperature (culture temperature), and maintains buoyancy in the gelled state.
Further, since the hysteresis is large, the sol-gel transition temperature can be widened, so that the material design is easy.

<Composition of culture solution>
The components of the culture solution are not particularly limited as long as they can have a phase transition temperature (Tc) during temperature increase and a phase transition temperature (Tcs) during temperature decrease. However, cellulose nanofibers and thermal sol gels having an average diameter of 2.0 nm to 100 nm It is desirable to include a modifier.

(Cellulose nanofibers having an average diameter of 2.0 nm to 100 nm)
The average diameter of the cellulose nanofiber (hereinafter referred to as “CNF”) is not particularly limited as long as it is 2.0 nm or more and 100 nm or less, but is preferably 3.0 nm to 50 nm, and more preferably 4.0 to 20 nm.
When the average diameter of CNF is less than 2.0 nm, the network structure of CNF is fragile, so even if the culture solution is in a gelled state, the suspension of the cells in culture is not sufficient and the cells tend to settle, so that good cells The growth rate cannot be achieved.
On the other hand, when the average diameter of CNF exceeds 100 nm, the network structure of CNF is strong, so even if the culture solution is in a sol state, the cultured cells are not sufficiently settled and difficult to settle. The sedimentation rate cannot be achieved.
In the present invention, after completion of the culture, it may indicate that the cells are settled simply by giving a temperature difference to the culture solution, and the recoverability may be good.

((Average diameter of CNF))
The average diameter of CNF can be adjusted by adjusting the manufacturing conditions.
For example, when producing CNF by mechanical crushing, the average diameter of CNF can be reduced as the pressure of the crusher is increased or the number of treatments (passes) of the crusher is increased. That is, the average diameter of CNF can be reduced. The average diameter of CNF can be increased as the pressure of the crusher is lowered or the number of treatments (pass times) of the crusher is decreased. That is, the average diameter of CNF can be increased.
In addition, for example, when CNF is produced by chemical cracking, chemical modification (for example, oxidation (see Japanese Patent No. 4998981), carboxymethylation (for example, see WO2015 / 109995), or phosphoric acid The average diameter of the CNF can be adjusted by mechanical disintegration after esterification (eg, see WO 2014/185505) and adjusting the disintegrator pressure and / or number of treatments. As in the case of producing CNF by mechanical crushing, the average diameter of CNF can be reduced as the pressure of the crusher is increased or the number of treatments (passes) of the crusher is increased. The average diameter of CNF can be increased as the pressure is decreased or the number of treatments (passes) of the crusher is decreased.

In addition, the average diameter of CNF was calculated | required with the following method (refer patent 5545453 specification).
A CNF aqueous dispersion diluted to have a CNF concentration of 0.001% by mass is prepared. This CNF dispersion is thinly spread on a mica sample stage and heated and dried at 50 ° C. to prepare an observation sample. The observation sample is observed using an AFM (Atomic Force Microscope), and the cross-sectional height of the observed shape image is measured at 10 points. The arithmetic average value of 10 measured values is taken as the average diameter of CNF.

((Average fiber length of CNF))
The average fiber length of CNF is not particularly limited, but is preferably 0.20 μm or more and 2.0 μm or less, more preferably 0.30 μm or more and 1.5 μm or less, and further preferably 0.40 μm or more and 1.0 μm or less. It is.
Within this range, when the culture solution is phase-shifted from the gelled state to the solated state, cells are more likely to settle and a better cell sedimentation rate can be achieved.
The average fiber length of CNF can be measured according to the method described in JP-T-2013-541956.

The average fiber length of CNF can be adjusted by adjusting the production conditions.
For example, when producing CNF by mechanical crushing, the average fiber length can be shortened as the temperature of the CNF dispersion during the crushing process is raised, and the average fiber length is lengthened as the temperature of the CNF dispersion is lowered. Can do.
In addition, for example, when CNF is produced by chemical cracking, chemical modification (for example, oxidation (see Japanese Patent No. 4998981), carboxymethylation (for example, see WO2015 / 109995), or phosphoric acid The average fiber length can be shortened as the treatment temperature is increased during esterification (for example, see International Publication No. 2014/185505), and the average fiber length can be lengthened as the treatment temperature is lowered.

((CNF content))
The higher the CNF content in the culture solution, the lower the temperature transition phase transition temperature (Tc), and the lower the CNF content, the higher the Tc.
The CNF content in the culture solution is not particularly limited, but is preferably 0.01% by mass or more and 1.0% by mass or less, more preferably 0.02% by mass or more and 0.50% by mass or less, more preferably It is 0.03 mass% or more and 0.10 mass% or less.

When the culture solution of the present invention contains CNF, it becomes easier to gel and the temperature transition phase transition temperature (Tc) shifts to a lower temperature side, so that it can be gelled at a lower temperature.
When the culture solution of the present invention contains CNF, gelation can be performed at a lower temperature than in the case where CNF is not included, and therefore, denaturation of medium components for culturing cells can be suppressed, which is preferable. When the CNF content is within the above range, the effect of lowering the temperature transition phase transition temperature (Tc) is more easily exhibited.
In addition, when the culture solution of the present invention contains CNF, it becomes easier to float cells in culture and it becomes more difficult to settle during culture, so that a better cell growth rate is obtained. Can be achieved.

((Carboxy group content))
CNF preferably contains a carboxy group.
The carboxy group content of CNF is not particularly limited, but is preferably 0.6 mmol / g or more and 2.0 mmol / g or less, more preferably 0.7 mmol / g or more and 1.9 mmol / g or less, and further preferably Is 0.9 mmol or more and 1.8 mmol / g or less.
When CNF contains a carboxy group, the dispersion of CNF is promoted by the carboxy group, the fibers of CNF spread widely in the culture medium of the present invention, and the interaction between CNFs increases. As a result, the effect of facilitating the sol-gel transition of the culture solution of the present invention is exhibited.
It also has the effect of increasing the interaction between CNF and the thermal sol-gel changing agent and promoting gel formation.

The method for introducing a carboxy group into CNF is not particularly limited. For example, TEMPO (2,2,6,6-tetramethyl-1-pyperizine-N-oxyl; 2,2,6,6-tetra Methyl-1-piperidine-N-oxyl) oxidation (see, for example, WO 2010/116826 or WO 2009/084566), and carboxymethylation (eg, WO 2014/116 No. 088072 or Japanese Patent No. 4055914). By adjusting the reaction time, the reaction temperature and / or the amount of the oxidizing agent during the oxidation treatment and / or the reaction time, the reaction temperature and / or the amount of the carboxymethylating agent during the carboxymethylation treatment, etc. The amount of group introduction can be adjusted.

In addition, the carboxy group content of CNF is determined by the following method (see Japanese Patent No. 5351586).
60 mL of a 0.5 mass% slurry of CNF is prepared, and 0.1 M hydrochloric acid aqueous solution is added to adjust the pH to 2.5. A 0.05 M aqueous sodium hydroxide solution is dropped into the slurry, and the electrical conductivity is measured until the pH becomes 11. From the amount A (mL) of sodium hydroxide aqueous solution consumed in the neutralization step of the weak acid where the change in electrical conductivity is gradual, it is calculated by the following formula.
Carboxy group content (mmol / g) = A (mL) × 0.05 (mol / L) / CNF mass (g)

((CNF oxidation treatment))
Moreover, it is preferable that CNF is oxidized.
Examples of the oxidation treatment include the TEMPO oxidation treatment described above.
That is, TEMPO-modified CNF is a CNF that contains a carboxy group and is subjected to oxidation treatment.

(Thermal sol-gel change agent)
Tcs can be controlled by using a thermal sol-gel modifier in combination with CNF.
The thermal sol-gel modifier promotes sol formation when the temperature is lowered. That is, it has the effect of sol-gelating the culture solution in a gelled state without lowering to a very low temperature, that is, a drop that increases the sol-gelation temperature.
As a result, when the culture solution is made into a sol and the cells are precipitated and collected, it is not necessary to lower the culture solution temperature to such a low temperature that the cells die, and the cell mortality can be suppressed.

((Types of thermal sol-gel change agent))
The thermal sol-gel changing agent is not particularly limited, and examples thereof include methyl cellulose (MC) and curdlan (CU), preferably MC or CU, and more preferably MC.

((Molecular weight or degree of polymerization of thermal sol-gel change agent))
The degree of polymerization of MC and CU is not particularly limited, but is preferably 100 to 10,000, more preferably 200 to 6000, and further preferably 300 to 4000.
Within this range, the effect of solification can be more easily expressed, and the viscosity of the culture is further reduced, so that a better cell sedimentation rate can be achieved.

MC is not particularly limited, and MCs having various molecular weights or polymerization degrees can be used.
MC (for example, methylcellulose # 400, manufactured by Nacalai Tesque) has a gelation temperature (Tc) of about 60 ° C. and a solation temperature (Tcs) of about 20 ° C.
As commercially available MC, for example, methyl cellulose # 100, methyl cellulose # 400, methyl cellulose # 1500, or methyl cellulose # 4000 (all manufactured by Nacalai Tesque; numbers after # indicate the degree of polymerization) can be used.

CU is not specifically limited, For example, what was purchased from Wako Pure Chemical (for biochemistry) or Kirin Kyowa Foods can be used.
CU (for example, curdlan (for biochemistry), manufactured by Wako Pure Chemical Industries, Ltd.) has a gelation temperature (Tc) of about 80 ° C. and a solation temperature (Tcs) of about 15 ° C.

((Thermal sol-gel changing agent content))
The higher the temperature of the thermal sol-gel modifier in the culture solution, the higher the temperature transition phase transition temperature (Tcs), and the lower the content of the thermal sol-gel, the lower Tcs.
That is, the thermal sol-gel modifier has the effect of increasing the temperature at which the culture solution is solated. As a result, when the culture solution is made into a sol and the cells are precipitated and collected, it is not necessary to lower the culture solution temperature to such a low temperature that the cells die, and the cell mortality can be suppressed.

The content of the thermal sol-gel changing agent in the culture solution of the present invention is not particularly limited, but is preferably 0.05% by mass or more and 3.0% by mass or less, more preferably 0.1% by mass or more and 1.5% by mass or less. It is not more than mass%, more preferably not less than 0.2 mass% and not more than 0.7 mass%.

If the content of the thermal sol-gel change agent is within this range, the effect of sol-solation of the thermal sol-gel change agent is better exhibited, Tcs does not become too low, and the temperature lowering treatment temperature of the cell does not become low. , Cell mortality can be further reduced. Moreover, since the difference between Tcs and Tc is large, the culture solution becomes more stable and the cell growth rate is more likely to be improved.

(Give sol-gel transition ability)
Preferred embodiments of the present invention have the following characteristics.
When the culture solution contains CNF and a thermal sol-gel changing agent, the culture solution is imparted with a sol-gel transition ability.
A high cell growth rate can be achieved by gelling the culture medium and subjecting the cells to suspension culture in the gelled culture medium.
After performing floating culture in the gel of the culture solution, the culture solution is made into a sol, and the cultured cells are sedimented to facilitate recovery.
Such gelation and solification are surprisingly manifested by the interaction between CNF and the thermal sol-gel modifier.
When the temperature of the culture solution is raised to a temperature equal to or higher than the gelation temperature (Tc), a hydrogel is formed in the culture solution to gel and the cells float, but when the temperature of the culture solution falls below the solation temperature (Tcs), The hydrogel collapses and the cells settle.
Although it is possible to express the gelation temperature (Tc) and the solation temperature (Tcs) only with the thermal sol-gel changing agent, it is desirable to use CNF in combination in order to ensure cell floating during culture.

(Adjustment of ΔTc)
Note that Tc can be adjusted by the content of CNF, and Tcs can be adjusted by the content of the thermal sol-gel modifier, and since these are independent, ΔTc = Tc−Tcs is CNF and thermal sol-gel. It can be adjusted by the content ratio of the changing agent.

(Medium components for culturing cells)
The culture solution of the present invention may contain a medium component for culturing cells.
The medium components for culturing cells can be appropriately selected depending on the cell type. Examples of the medium containing basal medium components for culturing human cells include, for example, Eagle's minimal essential medium (EMEM (Eagle's minimal essential) medium)) and / or modified media thereof. Examples of the modified medium of EMEM include Dulbecco's modified Eagle's modified Eagle medium (DMEM (Dulbecco's modified Eagle medium)). Moreover, as a culture medium containing a basal medium component, what added components, such as a vitamin, an amino acid, and glucose, to these minimum essential culture media is also contained.

<Temperature raising process>
The temperature raising step is a step of raising the temperature of the culture solution from below the gelation temperature Tc to above the solation temperature Tc.
In the temperature raising step, the culture solution is gelled by raising the temperature of the culture solution to Tc or higher by heat treatment.

The gelation temperature Tc can be appropriately adjusted by adjusting the components of the culture solution, for example, the content of cellulose nanofibers, and is not particularly limited, but is preferably 42.0 ° C or higher and 90.0 ° C or lower. More preferably, it is 44.0 degreeC or more and 85.0 degreeC or less, More preferably, it is 45.0 degreeC or more and 80.0 degreeC or less.
When the gelation temperature Tc is within this range, oxygen diffuses to the inside of the gelled culture solution in the culturing step, so that the cell growth rate can be further increased. In addition, since the medium components for culturing the cells are not decomposed and the cultured cells can be used, the cell growth rate can be further increased.

The temperature at which the culture solution is gelled, that is, the temperature of the heat treatment is not particularly limited as long as it is Tc or higher, but is preferably Tc or higher and 99.0 ° C or lower, more preferably Tc + 2.0 ° C or higher and 97.0 ° C. It is below, More preferably, it is Tc + 4.0 degreeC or more and 95.0 degreeC or less.
When the temperature of the heat treatment is within this range, the gelation of the culture solution proceeds sufficiently, and sufficient buoyancy is imparted to the cells in the culturing step, so that the cell growth rate is further improved. In addition, since the medium components for culturing the cells are not decomposed by the heat treatment, the cell growth rate is not lowered.
Since the lower limit of the temperature of the heat treatment depends on the gelation temperature (Tc), it is determined depending on the culture solution. On the other hand, the upper limit of the temperature of the heat treatment is defined by the thermal decomposition of the medium components for culturing cells, and therefore does not depend on the gelation temperature (Tc).

The time for the heat treatment is not particularly limited as long as the culture solution can be gelled at the heating temperature at that time, but is preferably 1.0 second or more and 10 minutes or less, more preferably 5.0 seconds. It is not less than 8.0 minutes and more preferably not less than 10 seconds and not more than 5.0 minutes.
When the heat treatment time is within this range, the gelation of the culture solution proceeds sufficiently, and sufficient buoyancy is imparted to the cells in the culturing step, so that the cell growth rate is further improved. In addition, since the medium components for culturing the cells are not decomposed by the heat treatment, the cell growth rate is not lowered.
In addition, among the medium components for culturing cells, components that are easily denatured by heating, such as amino acids, proteins, sugars, and vitamins, are not added in the culture solution preparation step described later, but are added to the culture solution after the temperature raising step. It is also preferable to add.

As described later, the cell culture method of the present invention may perform a seeding process in which cells are seeded in a culture solution. However, when the seeding process is performed before the temperature raising process, the temperature of the heat treatment in the temperature raising process. Is preferably 42.0 ° C or higher and 50.0 ° C or lower, more preferably 42.0 ° C or higher and 48.0 ° C or lower, and further preferably 42.0 ° C or higher and 45.0 ° C or lower.
When the temperature of the heat treatment is within this range, cell death due to heating can be suppressed, and the cell growth rate in the culture step can be improved.

《Heating method》
The method (heating method) for the heat treatment is not particularly limited, and examples thereof include a method in which the culture vessel is placed on a plate heater and the culture vessel is heated, and a method in which the culture vessel is heated with a hot water bath. Alternatively, a method using a radiant heat source (infrared heater or the like) may be used.

<Culture process>
The culture process is a process in which cells are suspended in the culture solution at a culture temperature of Tcs or higher after the culture solution is heated to Tc or higher in the temperature raising step.

In the temperature raising step, the culture solution is heated to a temperature equal to or higher than the gelation temperature (Tc) and gelled, and then cell suspension culture is performed.
After the culture solution is gelled, before the start of the culture, the culture solution is kept in a gelled state, preferably the temperature of the culture solution is maintained at the solation temperature (Tcs) or higher.
In addition, after the start of the culture, that is, during the culture, the temperature of the culture solution is maintained at Tcs or higher.
If the temperature of the culture solution is maintained at Tcs or higher after the culture solution is gelled, the culture solution tends to maintain a gelled state.
In addition, the temperature of the culture solution during culture, that is, the culture temperature will be described later.

During culture, as long as the temperature of the culture solution is maintained at Tcs or higher, the temperature may be increased or decreased within the range of Tcs or higher. This is because the gelled culture fluid has a thermodynamically stable gel structure, and thus maintains a stable gel structure at Tcs or higher.

As the culture method, for example, the methods described in International Publication No. 2015/111734 ([0032] to [0033]), International Publication No. 2014/136581 ([0063] to [0095]) may be used. it can.

<Culture temperature>
The culture solution of the present invention is characterized by the presence of hysteresis in the gelation temperature (Tc) and the solation temperature (Tcs), although sol-gel transition occurs due to heat.
Tc> Tcs, and as a result, the gel state can be maintained even when the temperature is lowered to a low temperature, so that the gelation temperature is widened and the culture temperature can be widely taken.

Therefore, the culture temperature is not particularly limited as long as it is Tcs or higher, but is preferably Tcs or more and Tc or less, more preferably Tcs + 2.0 ° C. or more and Tc−2.0 ° C. or less, and further preferably Tcs + 5.0 ° C. The temperature is Tc−5.0 ° C. or lower.

More specifically, the culture temperature is not particularly limited as long as the culture solution is in a gelled state, but is preferably 10.0 ° C or higher and 55.0 ° C or lower, more preferably 15.0 ° C or higher. It is 50.0 ° C. or lower, more preferably 20.0 ° C. or higher and 45.0 ° C. or lower.

"cell"
A cell is not specifically limited, A various cell can be included.
Although the origin of a cell is not specifically limited, Preferably it is an animal cell, More preferably, it is a human cell.
The cell type is not particularly limited, but is preferably a stem cell, more preferably an embryonic stem cell (hereinafter sometimes referred to as “ES cell”: ES, embryonic stem), somatic stem cell, and induced pluripotent stem cell. (Hereinafter, it may be referred to as “iPS cell.”: At least one selected from the group consisting of iPS, induced pluripotent stem).
The origin and type of the cells are not particularly limited, but are preferably human stem cells, more preferably human embryonic stem cells (human ES cells), human somatic stem cells, and human induced pluripotent cells (iPS cells). It is at least one selected from the group.
This is because stem cells are relatively resistant to temperature changes, and even if the temperature is changed from the culture temperature to the temperature of the temperature lowering treatment, the cell survival rate can be increased.

Examples of somatic stem cells include hematopoietic stem cells, umbilical cord blood stem cells, satellite cells, intestinal stem cells, hair follicle stem cells, mesenchymal stem cells, neural stem cells, endothelial stem cells, olfactory mucosal stem cells, neural crest stem cells, and testis cells. Preferably, it is a mesenchymal stem cell.
Examples of human somatic stem cells include human hematopoietic stem cells, human umbilical cord blood stem cells, human satellite cells, human intestinal stem cells, human hair follicle stem cells, human mesenchymal stem cells, human neural stem cells, human endothelial stem cells, human olfactory mucosa Examples include stem cells, human neural crest stem cells, and human testis cells, with human mesenchymal stem cells being preferred.

Mesenchymal stem cells that can differentiate into various types of cells and that can be used for treatment usually fall into the collection, selection, and purification from living organisms. The mesenchymal stem cells that can be used in the recovery method of the present invention include not only clinical primary human mesenchymal stem cells directly collected from patients, but also mesenchymal stem cells that can be obtained from a cell bank that can be used for test research, And immortalized mesenchymal stem cell lines.
As is known to those skilled in the art, these mesenchymal stem cells can be any cells derived from autologous sources, xenogeneic sources, or xenogeneic sources from the viewpoint of clinical application. Good. The collection source can also be any collection source such as donor bone marrow, tissue biopsy, embryonic source, or postnatal source. Specifically, bone marrow from the iliac crest, femoral neck, spine, ribs, or other bone marrow cavities, or embryonic yolk sac, placenta, umbilical cord, periosteum, fetal or adolescent skin, and tissue life including blood Examples include collection sources such as laboratory tests.

Examples of iPS cells include, for example, International Publication No. 2015/037535, Japanese Patent No. 5590646, International Publication No. 2011/043405, International Publication No. 2013/0777423, or International Publication No. 2014/136581 ([0050] to [0061]) can be used.

In addition, “cultured cells” in the sedimentation process, “seeded cells” in the seeding process described later and “cells” as seed cells, and “cells to be cultured in suspension” in the culture process described later and floating from the seed cells Any of the “cells” to be cultured may be the cells described above.

<Sowing process>
In the cell culture method of the present invention, the seeding step may be performed before or after the temperature raising step.
The seeding process is a process of seeding cells in a culture solution.
The method of seeding the cells in the culture solution is not particularly limited, and examples thereof include a method of injecting a suspension prepared by dispersing cells in the culture solution into the culture solution.

《Seeding timing》
The seeding step may be performed after the temperature raising step (sometimes referred to as “ART” (after rising temperature)) or may be referred to as before the temperature raising step (“BRT” (before rising temperature)). ).

《Cell concentration at seeding》
In the seeding step, the cell concentration at the time of seeding is not particularly limited, but is preferably 1.0 × 10 ⁵ cells / mL to 1.0 × 10 ¹⁰ cells / mL, more preferably 1.0 × 10 ^6. The number per unit / mL to 1.0 × 10 ⁹ units / mL, and more preferably 1.5 × 10 ⁶ units / mL to 5.0 × 10 ⁸ units / mL.

<Seeding process after gelation>
In the cell culture method of the present invention, the seeding step performed after the temperature raising step is particularly referred to as a seeding step after gelation.
When performing the seeding step after gelation, the cells can be diffused to the inside of the medium by gently stirring the culture solution in the gelled state and seeding the cells. Examples of the method of stirring the culture solution include a method of stirring manually using a spatula, a method of stirring using a magnetic stirrer, and a stirring bar. The number of rotations during stirring is not particularly limited, but is preferably 10 rpm to 150 rpm, and the stirring time is not particularly limited, but is preferably 0.1 minute to 5 minutes. This is due to the following reason.
When the culture solution of the present invention is in a gelled state, the gel structure is thermodynamically stable, so even if the gel structure is broken by stirring, it can be easily reconstructed if it is in a temperature range that can maintain the gelled state. Is done. The time until reconstruction is longer as the degree of destruction of the gel structure is larger. However, weak structural destruction by weak agitation restores the structure in 1 to 30 minutes and enables cell suspension culture.
Furthermore, the agitation may be performed before sowing or after sowing. Because of weak agitation, unlike strong agitation such as centrifugation, cells do not die even after seeding.
In the seeding step after gelation, the temperature of the culture solution was raised to Tc ° C. or higher in the temperature raising step to cause gelation, and the temperature of the culture solution was preferably kept at Tcs or higher while keeping the culture solution in a gelled state. It is done as it is.
If the temperature of the culture solution is maintained at Tcs or higher after the culture solution is gelled, the culture solution tends to maintain a gelled state.

(Seeding process after gelation-temperature of the culture solution at the time of sowing)
In the seeding step after gelation, the temperature of the culture solution when seeding cells is not particularly limited as long as it is equal to or higher than the solation temperature Tcs, but is preferably Tcs or higher and Tc or lower, more preferably Tcs + 2.0 ° C or higher. Tc−2.0 ° C. or lower, more preferably Tcs + 5.0 ° C. or higher and Tc−5.0 ° C. or lower.
The temperature of the medium at the time of seeding is preferably around the culture temperature in order to reduce the stress applied to the cells.

<< Sowing process before gelation >>
In the cell culture method of the present invention, the seeding step performed before the temperature raising step is particularly referred to as a pre-gelation seeding step.
In the case of performing the pre-gelation seeding step, it is desirable that the temperature transition phase transition temperature (Tc) is low. Even if the temperature raising step is performed after the seeding step, the culture solution can be gelled at a low temperature, so that cell death associated with the heat treatment in the temperature raising step can be suppressed, and the cell growth rate after cultivation is increased. And / or lower cell mortality after sedimentation.
Such a temperature transition phase transition temperature (Tc) is affected by the heat resistance of cells, the survival rate threshold, and the like and cannot be defined uniformly, but is preferably 45.0 ° C. or less, more preferably 43 It is 0.0 degrees C or less, More preferably, it is 42.0 degrees C or less.

(Pre-gelation sowing step-temperature of the culture solution at the time of sowing)
In the seeding step before gelation, the temperature of the culture solution when seeding cells is not particularly limited as long as it is lower than the gelation temperature Tc, but is preferably Tcs or more and less than Tc, more preferably Tcs + 2.0 ° C. or more. Tc−2.0 ° C. or lower, more preferably Tcs + 5.0 ° C. or higher and Tc−5.0 ° C. or lower.
The temperature of the medium at the time of seeding is preferably around the culture temperature in order to reduce the stress applied to the cells.

<Recovery process>
In the cell culture method of the present invention, a collection step may be further performed after the culture step.
The recovery step is a step of lowering the temperature of the culture solution below Tcs, precipitating and recovering the cells cultured in the culture step.
A process for lowering the temperature of the culture solution below Tcs is called a temperature lowering process, and the operation may be called a temperature lowering operation.
In the present specification, Tcs may be referred to as a solubilization temperature. However, from the definition of Tcs, the culture solution is in a gelled state at Tcs.

The solubilization temperature Tcs can be appropriately adjusted by adjusting the components of the culture solution, for example, the content of the thermal sol-gel changing agent, but is not particularly limited, but is preferably 3.0 ° C. or higher and 41.0 ° C. or lower. More preferably, it is 5.0 degreeC or more and 40.0 degreeC or less, More preferably, it is 10.0 degreeC or more and 38.0 degreeC or less.
When the solation temperature Tcs is within this range, cells can be allowed to settle in the recovery process without lowering the temperature, and the transition from the gelled state to the solated state is stabilized. Can be further reduced, and the cell sedimentation rate can be further improved.

In the collection step of the cell culturing method of the present invention, in order to precipitate the cells cultured in the culturing step, it is only necessary to lower the temperature of the culture solution below Tcs. By lowering the temperature of the culture solution to a temperature lower than Tcs (hereinafter sometimes referred to as “temperature reduction treatment temperature”), the culture solution in a gelled state is made into a sol, and the cultured cells are allowed to settle. Can be recovered.

The temperature of the temperature lowering treatment is not particularly limited as long as it is lower than Tcs, but is preferably 3.0 ° C or higher and Tcs-3.0 ° C or lower, more preferably 5.0 ° C or higher and Tcs-5.0 ° C or lower. .
When the temperature-lowering treatment temperature is within this range, sedimentation is sufficient, the cell recovery rate is further improved, the temperature stress applied to the cells is small, and the cell mortality rate is further reduced.
Since the upper limit of the temperature lowering treatment depends on the solubilization temperature (Tcs), it is determined depending on the culture solution. On the other hand, since the lower limit of the temperature lowering process depends on the cell viability at that temperature, it does not depend on the solubilization temperature (Tcs).

The time for the temperature lowering treatment, that is, the time for keeping the culture solution at the temperature for the temperature lowering treatment is not particularly limited as long as the culture solution can be made into a sol, but is preferably 1 second or more, more preferably 10 seconds or more. More preferably, it is 30 seconds or more.

《Cooling method》
The method for cooling treatment (cooling method) is not particularly limited, and examples thereof include a method of immersing the incubator in a refrigerant such as water and a method of bringing a cooler into contact with the incubator. The temperature may be lowered by applying cold air.

《Recovery method》
After the culture solution is solated and the cells are precipitated, the cells can be recovered by removing the supernatant culture solution by a method such as decantation, decant aspiration, or aspiration.

<Culture solution preparation process>
In the cell culturing method of the present invention, a culture solution preparation step may be first performed.
The culture solution preparation step is a step of preparing a culture solution for culturing cells.
When the culture solution of the present invention contains CNF and a thermal sol-gel change agent, the culture solution preparation step can include two stages of preparation of CNF and preparation of the culture solution.
However, when an off-the-shelf product is used as CNF, preparation of CNF can be omitted.

<< Preparation of CNF >>
(1) Preparation method of TEMPO (2,2,6,6-tetramethyl-1-pyperizine-N-oxyl; 2,2,6,6-tetramethyl-1-piperidine-N-oxyl) CNF TEMPO CNF The preparation method is not particularly limited. For example, according to the method described in JP-A-2009-263853 ([0015] to [0030]), the cellulosic material is present in the presence of an oxidizing agent (sodium hypochlorite). Below, TEMPO (2,2,6,6-tetramethyl-1-pyperizine-N-oxyl; 2,2,6,6-tetramethyl-1-piperidine-N-oxyl) catalyzed oxidation treatment is performed, and then oxidized. A TEMPO-modified cellulose nanofiber can be prepared by subjecting the cellulose-based raw material to a wet atomization treatment using an ultra-high pressure homogenizer and defibrating.

(2) Method for preparing CM (carboxymethyl) CNF The method for preparing CM CNF is not particularly limited. For example, according to the method described in International Publication No. 2015/109995 ([0056]), Preparation of CMized cellulose nanofibers by pulverizing the cellulose raw material that has been CMized with monochloroacetic acid, a CM agent, in the presence of an alkali catalyst by wet atomization using a high-pressure homogenizer can do.
In addition, JP2015-227517A, JP2015-134873A, JP2015-4032A, JP2014-193580A, JP2013-185122A, JP3642147A, JP4055914A. Gazette, International Publication No. 13/137140, International Publication No. 2015/107995, International Publication No. 2015/50117, International Publication No. 2014/181560, International Publication No. 2014/181260, International Publication No. 2014/088072. Alternatively, the preparation method of C-converted CNF described in International Publication No. 2014/087767 can be used.

(3) Phosphate-treated CNF
It can be prepared by introducing a phosphate group into CNF, more preferably by phosphoric acid esterification.
For example, the methods described in International Publication No. 2014/185505, Japanese Patent Application Laid-Open No. 2016-37031, International Publication No. 2016/002689, or International Publication No. 2016/002688 can be used.

(4) Method for preparing mechanically crushed CNF The method for preparing mechanically crushed CNF is not particularly limited. For example, according to the method described in International Publication No. 2015/111734 ([0039]), a cellulosic material is used. The mechanically crushed CNF can be prepared by wet atomization using a high-pressure homogenizer and defibrating.

In the preparation of various CNFs, the origin of the raw material CNF is not particularly limited, and any of CNF derived from pulp, CNF derived from bacterial cellulose (BC), and nanofibers by electrospinning can be used. Preferably, it is CNF derived from pulp. This is because the fiber strength is strong and the spheroids are difficult to subdivide during passage.

When selected from TEMPO CNF, CM CNF, and mechanically crushed CNF, TEMPO CNF or CM CNF is preferred, and TEMPO CNF is more preferred.
That is, the CNF is preferably CNF containing a carboxy group, more preferably CNF containing a carboxy group and subjected to oxidation treatment.
The TEMPO-modified CNF is introduced with a carboxy group and subjected to an oxidation treatment by a TEMPO oxidation treatment.
C-converted CNF has a carboxy group introduced by carboxymethylation but has not been subjected to oxidation treatment.
Mechanically crushed CNF has no carboxy group introduced and is not oxidized.

<Preparation of culture solution>
(Preparation of CNF-free culture solution)
A basal medium suitable for the cells to be used can be prepared by a conventionally known method.
For example, a medium described in International Publication No. 2015/111734 ([0029] to [0031]) or a medium described in International Publication No. 2014/136581 ([0062]) may be used.

(Addition of CNF and thermal sol-gel modifier)
By adding CNF and a thermal sol-gel change agent to the basal medium, a culture solution used in the cell culture method of the present invention is prepared.
The order of addition of CNF and the thermal sol-gel change agent is not particularly limited, and the thermal sol-gel change agent may be added after CNF is added, or the thermal sol-gel change agent may be added before CNF is added. Moreover, CNF and a thermal sol-gel changing agent may be simultaneously added to the basal medium.
When CNF is added after the addition of the thermal sol-gel changing agent, the agitation is performed when CNF is added to promote mixing of CNF and the thermal sol-gel changing agent, and due to the interaction between CNF and the thermal sol-gel changing agent. A sol-gel transition can be easily caused.
In addition, when the thermal sol-gel changing agent is added after the addition of CNF, rapid mixing is performed when the thermal sol-gel changing agent is added to promote mixing of the thermal sol-gel changing agent and CNF. The sol-gel transition due to the interaction with can be easily caused.
Furthermore, when CNF and a thermal sol-gel change agent are added simultaneously, rapid mixing is performed when adding CNF and a thermal sol-gel change agent to promote mixing of the CNF and the thermal sol-gel change agent, thereby changing the CNF and the thermal sol-gel change agent. The sol-gel transition due to the interaction with the agent can be easily caused.
Specifically, when adding CNF and / or a thermal sol-gel change agent to a culture medium, it is preferable to stir with a magnetic stirrer or the like, and it is preferable to stir with a high-speed homogenizer after the addition.
The rotation speed of the homogenizer is preferably 1000 rpm to 100,000 rpm, more preferably 3000 rpm to 50,000 rpm, and still more preferably 6000 rpm to 20,000 rpm.
The stirring time of the homogenizer is preferably 10 seconds to 30 minutes, more preferably 20 seconds to 15 minutes, and further preferably 30 seconds to 10 minutes.
When the rotation speed and time are both within this range, both the cell growth rate and the cell sedimentation rate are likely to be improved.

After adding the thermal sol-gel changing agent, it is desirable that the temperature of the culture solution does not become higher than the temperature transition phase transition temperature (Tc) until the temperature is raised in the temperature raising step described above. This is because the gel state has a too high viscosity, and the handleability is worse than the sol state.

The culture medium is based on the medium described in International Publication No. 2015/111734 ([0029] to [0031]) or the medium described in International Publication No. 2014/136581 ([0062]). Can also be used.

The amount of the culture solution per culture vessel is not particularly limited, but is preferably 0.5 L or more, more preferably 0.6 L or more and 50 L or less, and further preferably 0.7 L or more and 5.0 L or less.
When the amount of the culture solution is within this range, oxygen can be sufficiently supplied to the cells during suspension culture, so that the cell growth rate can be increased and a large number of cells can be cultured. As a result, it becomes easy to efficiently collect the cultured cells.

[Culture medium]
The present invention also provides a culture solution for use in the cell culture method of the present invention.
The culture solution is as described above.

[Examples 1 to 6, Comparative Example 1]
Examples 1 to 6 and Comparative Example 1 are examples showing the effect of the thermal sol-gel changing agent.

<Comparative example 1>
Comparative Example 1 is an example in which a culture solution containing cellulose nanofibers but not containing a thermal sol-gel changing agent was used.

<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
According to the method described in JP-A-2009-263853 ([0015] to [0030]), a cellulose-based material is treated with TEMPO (2,2,6,6-) in the presence of an oxidizing agent (sodium hypochlorite). tetramethyl-1-pyperizine-N-oxyl; 2,2,6,6-tetramethyl-1-piperidine-N-oxyl) Using a super high pressure homogenizer, the cellulose raw material oxidized by catalytic oxidation A TEMPO-modified cellulose nanofiber was prepared by defibrating by wet atomization.
When the average diameter and carboxy group introduction amount of the prepared TEMPO-modified cellulose nanofiber were measured according to the method described later, the average diameter was 4.0 nm and the carboxy group introduction amount was 1.7 mmol / g. Hereinafter, this TEMPO-modified cellulose nanofiber may be referred to as “TEMPO1”.

Measurement Method of Average Diameter A cellulose nanofiber aqueous dispersion diluted to have a cellulose nanofiber concentration of 0.001% by mass (hereinafter sometimes referred to as “CNF aqueous dispersion”) was prepared. This CNF aqueous dispersion is thinly spread on a mica sample stage, heated and dried at 50 ° C. to prepare a sample for observation, and the cross-sectional height of the shape image observed using an atomic force microscope (AFM) 10 points were measured, and the average diameter was calculated.

・ Measurement method of introduction amount of carboxy group 60 mL of a 0.5% by mass slurry of cellulose nanofibers was prepared and adjusted to pH 2.5 by adding 0.1 M hydrochloric acid aqueous solution, and then 0.05 M sodium hydroxide aqueous solution was added dropwise to adjust the pH. Was measured using the following formula from the amount (a) of 0.05 M aqueous sodium hydroxide solution consumed in the neutralization step of the weak acid where the change in electrical conductivity was mild. .
Carboxy group introduction amount (mmol / g) = a (mL) × 0.05 / cellulose nanofiber mass (g)

(Preparation of culture solution)
The prepared TEMPO1 was mixed with pure water to obtain a 0.3 mass% aqueous dispersion (hereinafter sometimes referred to as “CNF dispersion”). This CNF dispersion was autoclaved at 121 ° C. for 15 minutes, cooled to room temperature after sterilization.
Water is added to a powder medium (Dulbecco Modified Eagle Medium 2, manufactured by Nissui Pharmaceutical Co., Ltd.) and stirred with a magnetic stirrer for about 30 minutes, and Dulbecco Modified Eagle Medium 2 solution (hereinafter sometimes referred to as “DMEM2 solution”). Was prepared. The DMEM2 solution was autoclaved at 121 ° C. for 15 minutes, cooled to room temperature after sterilization.
After the autoclave sterilization, the sterilized CNF dispersion was dropped into the DMEM2 solution cooled to room temperature under aseptic conditions so as to have the concentrations shown in Table 1. After dropping, the DMEM2 solution to which the CNF dispersion was added was stirred at 10000 rpm for 5 minutes using a homomixer to obtain a CNF-added DMEM2 solution.
After cooling the CNF-added DMEM2 solution to room temperature, a filter-sterilized L-glutamine solution (L-glutamine solution (× 100), manufactured by Wako Pure Chemical Industries, Ltd .; 200 mmol / L) is adjusted to a final concentration of 2 mM. Added. Further, 1 mL of a sterilized 10% by mass aqueous sodium hydrogen carbonate solution was added to obtain an amino acid-added DMEM2 solution.
A small amount of the obtained amino acid-added DMEM2 solution was collected, and it was confirmed that the pH of the solution was within the range of pH 7.0 to 8.0 using pH test paper. FBS (fetal bovine serum) was added to the amino acid-added DMEM2 solution remaining after collecting a small amount so as to be 10% by volume before use to obtain a culture solution used for cell culture.
As a culture vessel, a hydrophilic polystyrene-treated culture vessel (floating cell culture flask MS-21800, manufactured by Sumitomo Bakelite Co., Ltd .; culture area 225 cm ² ; capacity 800 mL) was prepared. As the culture vessel, a culture vessel having a culture area (bottom area) in which the height of the culture solution when the culture solution was injected was 4.0 to 5.0 cm was selected.
The prepared culture solution was aseptically injected into the prepared culture container in the amount shown in Table 1.

Moreover, the viscosity of the prepared culture solution was measured.
(1) Measurement of viscosity during temperature rise While the temperature of the prepared culture solution is raised from 3.0 ° C to 98.0 ° C at a rate of temperature rise of 5.0 ° C / min, a plate viscometer (MCR301, Anton) The viscosity was measured under the measurement conditions of plate diameter = 45 mm, gap = 0.049 mm, and shear rate = 0.1 s ⁻¹ . In the range from 3.0 ° C. to 98.0 ° C., the viscosity did not become 10 times the viscosity at 3.0 ° C.
(2) Measurement of viscosity during temperature reduction The viscosity of the culture solution heated to 98.0 ° C was measured under the same measurement conditions as when the temperature was increased to 3.0 ° C while the temperature was decreased at a temperature decrease rate of 5.0 ° C / min. In the range from 98.0 ° C. to 3.0 ° C., the viscosity did not become 10 times the viscosity at 3.0 ° C.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution poured into the culture vessel was subjected to heat treatment at the temperature described in the “temperature of heat treatment” column of “temperature raising step” in Table 1 for the time described in the “time of heat treatment” column.

<< Seeding process >>
Human mesenchymal stem cells (hMSC (human mesenchymal stem cells), manufactured by Lonza; catalog number PT-2501) (hereinafter sometimes referred to as “hMSC”) are placed in the culture container into which the culture solution has been injected. Inoculated so that the cell concentration described in “Cell concentration at seeding [cells / mL]” in “Seeding step” (seed cell seeded in the culture solution in the culture vessel is referred to as “seeded cell dispersion solution” below). "). Specifically, a suspension prepared by dispersing human mesenchymal stem cells PT-2501 in a culture solution (hereinafter referred to as “PT-2501 suspension”) was injected into the culture solution.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 1.

<< Culture process >>
After the temperature raising step, the culture vessel in which the cells were seeded was set to the temperature described in the column of “Average temperature of culture medium” in “Culture step” in Table 1 (Heracoll VIOS CO ₂ incubator, Thermo Fisher stock) The culture of the cells was started (the cell dispersion liquid consisting of the cells in suspension culture and the culture liquid in the culture process is referred to as “floating cell dispersion liquid”).
The culture conditions were a carbon dioxide concentration of 5.0% by volume and a humidity of 95% RH.
One day after the start of the culture, in order to remove cells adhering to the culture container, the suspended cell dispersion in the culture container was transferred to a new Aznol sterilization container (Aznol sterilization dish GD90-15, manufactured by Azwan), and further cultured for 4 days. Continuously, spheroids were formed.

<< Recovery process >>
After completion of the culture, after exposure for 5 minutes to the temperature described in “Temperature of temperature reduction treatment” in “Recovery step” of Table 1, the means described in the “Sedimentation means” column of “Recovery step” in Table 1, Cultured cells were allowed to settle.
Total number of cells, cell growth rate, cell sedimentation rate, and cell mortality in cultured cell dispersion (referred to as a cell dispersion consisting of cultured cells and culture in a culture vessel that has been cultured in the collection step) Was determined according to the method described below.

-Method for calculating the total number of cells After 2 mL of the cultured cell dispersion was sampled and the cells forming spheroids were separated by trypsin treatment, the number of cells per 1 mL of the cultured cell dispersion was measured using an optical microscope. This was multiplied by the amount of the cultured cell dispersion to calculate the total number of cells in the cultured cell dispersion.

-Calculation method of cell growth rate The cell growth rate was calculated by the following formula, assuming that the total number of cells in the cultured cell dispersion was N and the number of seeded cells was Nf.
Cell growth rate = N / Nf times The higher the cell growth rate, the better.

・ Calculation method of cell sedimentation rate 1) Measurement of total spheroid count (N) before cooling operation:
2 mL of the cultured cell dispersion was sampled and injected into a polystyrene culture well having a diameter of 20 mm maintained at 37 ° C. The central portion of the culture well was observed with 10 optical fields at a magnification of 200 using an optical microscope, and the number of spheroids was measured for each visual field. The average value was N _1. This was repeated with respect to the culture solution of 10 culture wells, and N ₂ , N ₃ ,..., N ₁₀ were obtained. From the average value of N ₁ to N _10, the number of spheroids per mL of cultured cell dispersion N / mL was calculated. A cell mass having a diameter of 50 μm or more was treated as a spheroid.
2) Measurement of the number of floating (non-sedimenting) spheroids (n) after the temperature lowering operation:
After the temperature of the culture well was lowered, the supernatant of the culture solution in the well was gently collected. The collected supernatant was similarly observed using a microscope at 10 magnifications at a magnification of 200 times, and the number of floating spheroids in each visual field was measured. This average value was defined as n1. This was repeatedly measured with respect to the culture solution of ten culture wells, and n ₂ , n ₃ ,..., N ₁₀ were obtained. From the average value of n ₁ to n _10, the number of floating spheroids per mL of supernatant was calculated n 1 / mL. A cell mass having a diameter of 50 μm or more was treated as a spheroid.
3) Calculation of cell sedimentation rate:
The cell sedimentation rate (%) was calculated by the following formula.
Cell sedimentation rate (%) = {(N−n) / N} × 100 (%)
The higher the cell sedimentation rate, the better, and it is particularly desirable that the cell sedimentation rate is 75.0% or more.

Method for calculating cell mortality For cells before the temperature lowering operation, according to the method described in JP-T-2013-541956 ([0082]), live cells and dead cells are dyed separately, and cell survival rate Xa before the temperature lowering operation is calculated. (%) Was calculated.
Next, the cell survival rate Xb (%) after the temperature lowering operation was calculated in the same manner for the cells after the temperature lowering operation.
The cell mortality due to the temperature lowering operation was calculated by the following formula.
Cell death rate (%) = Xa (%) − Xb (%)
The lower the cell mortality, the better, and it is particularly desirable that the cell mortality is 11.0% or less.

Furthermore, from the cell growth rate (a), the cell sedimentation rate (b), and the cell mortality rate (c), a score was calculated by the following formula to obtain a comprehensive evaluation.
Score = a × {(100−c) / 100} × (b / 100)
The higher the score, the better, especially 4.4 or higher.

As a result, as shown in Table 1, the cell growth rate was 8.2 times, the cell sedimentation rate was 35.0%, the cell death rate was 4.0%, and the score was 2.8.

<Example 1>
<< Culture solution preparation process >>
After cooling the CNF-added DMEM2 solution to room temperature, methylcellulose (methylcellulose 400, manufactured by Wako Pure Chemical Industries, Ltd.) is aseptically added to the concentration shown in Table 1, and then filtered and sterilized L-glutamine solution ( The point that L-glutamine solution (× 100), manufactured by Wako Pure Chemical Industries, Ltd .; 200 mmol / L) was added to a final concentration of 2 mM, and the series of operations from the addition of methylcellulose and subsequent operations were about 37 The same procedure as in Comparative Example 1 was performed except that the procedure was carried out at a temperature of ° C.
Furthermore, the phase transition temperatures (Tc and Tcs) of the prepared culture broth were measured by the following measuring method.

Measurement method of phase transition temperature of culture solution (1) Measurement of phase transition temperature (Tc) during temperature increase The temperature of the prepared culture solution is 3.0 ° C to 98.0 ° C and the temperature increase rate is 5.0 ° C / min. The temperature was measured with a plate viscometer (MCR301, manufactured by Anton Paar) while measuring the viscosity under the measurement conditions of plate diameter = 45 mm, gap = 0.049 mm, shear rate = 0.1 s ⁻¹ , The temperature at which the viscosity became 10 times the viscosity at 3.0 ° C. was defined as the “temperature transition phase transition temperature (Tc)”.
(2) Measurement of phase transition temperature (Tcs) at the time of temperature decrease The temperature was increased to 98.0 ° C., while the temperature was decreased to 5.0 ° C./min. Viscosity was measured at, and the temperature when the viscosity became 10 times the viscosity at 3.0 ° C. at the time of temperature rise was defined as “temperature transition phase transition temperature (Tcs)”.

<< Seeding process >>
After the temperature raising step, a seeding step was performed in the same manner as in Example 1.

<< Culture process >>
After the seeding process, the culture process was performed in the same manner as in Example 1.

<< Recovery process >>
After the culturing step, the recovery step was performed in the same manner as in Example 1 except that the temperature lowering treatment temperature was set to the temperature described in the column “Temperature lowering treatment temperature ° C.” in Table 1 “Recovery step”. It was.

As a result, as shown in Table 1, the cell growth rate was 8.9 times, the cell sedimentation rate was 87.0%, the cell death rate was 11.0%, and the score was 6.9.

<Example 2>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methyl cellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 1.
The prepared culture solution was injected into the culture container in the amount shown in Table 1 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
After the culture solution preparation step, the culture and the culture were carried out in the same manner as in Example 1 except that the temperature and time were set to the temperatures described in the “Temperature of heat treatment” column of “Temperature raising step” in Table 1. The culture solution injected into the container was heat-treated.

As a result, as shown in Table 1, the cell growth rate was 9.4 times, the cell sedimentation rate was 91.0%, the cell death rate was 5.0%, and the score was 8.1.

<Example 3>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methyl cellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 1.
The prepared culture solution was injected into the culture container in the amount shown in Table 1 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 1, the cell growth rate was 9.6 times, the cell sedimentation rate was 95.0%, the cell death rate was 4.0%, and the score was 8.8.

<Example 4>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methyl cellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 1.
The prepared culture solution was injected into the culture container in the amount shown in Table 1 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 1, the cell growth rate was 9.3 times, the cell sedimentation rate was 90.0%, the cell death rate was 3.0%, and the score was 8.1.

<Example 5>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methyl cellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 1.
The prepared culture solution was injected into the culture container in the amount shown in Table 1 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 1, the cell growth rate was 8.5 times, the cell sedimentation rate was 83.0%, the cell death rate was 8.0%, and the score was 6.5.

<Example 6>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methyl cellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 1.
The prepared culture solution was injected into the culture container in the amount shown in Table 1 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 1, the cell growth rate was 7.5 times, the cell sedimentation rate was 80.0%, the cell death rate was 8.0%, and the score was 5.5.

<Description of Results of Examples 1 to 6 and Comparative Example 1>
Examples 1 to 6 are examples in which a thermal sol-gel change agent (MC) was added.
Comparative Example 1 containing no MC does not have the solubilization temperature Tcs and the gelation temperature Tc, and the cell sedimentation rate is low (35.0%) because the cells hardly settle when the temperature of the culture solution is lowered.
The Tc of the culture solution depends on the CNF content, and the Tcs depends on the MC content. In Examples 1 to 6, the CNF content is the same and only the MC content is different.
Therefore, as the MC content increases, Tcs increases and ΔTc = Tc−Tcs decreases. As the MC content decreases, Tcs decreases and ΔTc increases. It can be seen that the larger the ΔTc, the better the cell growth rate.
In Example 1, the cell mortality rate is as high as 11.0%, which is considered to be because the temperature of the temperature lowering process was low in the recovery process.
Moreover, although the cell growth rate of Example 6 is as low as 7.5 times, this is because in the culture step, the gelation temperature Tc and the solation temperature Tcs are close, and the gelation state of the culture solution is unstable. It is thought that.

[Examples 7 to 9]
Examples 7 to 9 are examples showing the effect of the temperature of the heat treatment.

<Example 7>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
In accordance with the method described in International Publication No. 2015/107995 ([0056]), in the presence of an alkali catalyst, CM is obtained using monochloroacetic acid which is a carboxymethylation (hereinafter sometimes referred to as “CM conversion”) agent. The cellulose raw material subjected to the chemical treatment was subjected to wet atomization using a high-pressure homogenizer and fibrillated to prepare CM-modified cellulose nanofibers (hereinafter sometimes referred to as “CM1”). The average diameter and carboxy group introduction amount of the prepared CM1 were measured in the same manner as in Comparative Example 1, and as shown in Table 2.

(Preparation of culture solution)
As in Example 1, except that instead of TEMPO1, the prepared CM1 was used in the addition amount shown in Table 2, and methyl cellulose (MC) was used as the thermal sol-gel change agent in the addition amount shown in Table 2. Thus, a culture solution was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution injected into the culture vessel was subjected to heat treatment at the temperature described in the “temperature of heat treatment” column of the “temperature raising step” in Table 2 for the time described in the “time of heat treatment” column.

<< Seeding process >>
Instead of human mesenchymal stem cell PT-2501 (hMSC), human iPS cell IMR90-1 (WiCell Research Institute, Inc., Madison, Wis., USA) (hereinafter sometimes referred to as “IMR90-1”) is used. Cells were seeded in the same manner as in Example 1 except that the cell concentration at the time of seeding was the cell concentration shown in Table 2 and the temperature of the culture solution at the time of seeding was the temperature shown in Table 2. did.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 2.

<< Culture process >>
After the sowing step, the culturing step was carried out in the same manner as in Example 1 except that an incubator set to the temperature described in the column of “Average temperature of culture broth” in Table 2 was used.

<< Recovery process >>
After the culturing step, the recovery step was performed in the same manner as in Example 1 except that the temperature of the temperature lowering treatment was set to the temperature described in the column of “Temperature of the temperature lowering treatment” in Table 2 “Recovery step”. It was.

As a result, as shown in Table 2, the cell growth rate was 8.2 times, the cell sedimentation rate was 93.0%, the cell death rate was 4.0%, and the score was 7.3.

<Example 8>
<Culture solution preparation process>
A culture solution was prepared in the same manner as in Example 7.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution in the culture vessel was subjected to a heat treatment at the temperature described in the “temperature of heat treatment” column of the “temperature raising step” in Table 2 for the time described in the “time of heat treatment” column.

<< Seeding process >>
After the temperature raising step, a seeding step was performed in the same manner as in Example 7.

<< Culture process >>
After the seeding process, the culture process was performed in the same manner as in Example 7.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 7.

As a result, as shown in Table 2, the cell growth rate was 9.8 times, the cell sedimentation rate was 97.0%, the cell death rate was 3.0%, and the score was 9.2.

<Example 9>
<Culture solution preparation process>
A culture solution was prepared in the same manner as in Example 7.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
After the culture solution preparation step, the temperature was poured into the culture vessel in the same manner as in Example 7 except that the temperature was set to the temperature described in the column “Temperature of heat treatment” in “Temperature raising step” in Table 2. The obtained culture broth was heat-treated.

As a result, as shown in Table 2, the cell growth rate was 8.3 times, the cell sedimentation rate was 75.0%, the cell mortality rate was 4.0%, and the score was 6.0.

<Description of the results of Examples 7 to 9>
In Example 9, the cell growth rate was 8.3 times and the cell sedimentation rate was 75.0%, which was lower than the cell growth rate and cell sedimentation rate of Example 8.
This is considered to be because the temperature of the heat treatment in the temperature raising step was high and the gel strength was high in the culture step and the recovery step.
In Example 7, the cell growth rate was 8.2 times, which was lower than the cell growth rate of Example 8.
This is considered to be because buoyancy was reduced in the culture process because the temperature of the heat treatment in the temperature raising process was low and the gel strength was low.

[Examples 10 to 12]
Examples 10 to 12 are examples showing the effect of temperature of the temperature lowering process.

<Example 10>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
TEMPO-modified cellulose nanofibers (hereinafter “TEMPO2”) were prepared in the same manner as in Comparative Example 1 except that the amount of the oxidizing agent used and the pressure of the ultrahigh pressure homogenizer were adjusted so as to obtain the average diameter and carboxy group content shown in Table 2. Was prepared in some cases. The average diameter and carboxy group introduction amount of the prepared TEMPO 2 were measured in the same manner as in Comparative Example 1, and as shown in Table 2.

(Preparation of culture solution)
The point that the prepared TEMPO2 was used in the addition amount shown in Table 2 instead of TEMPO1, and curdlan (curdlan (for biochemistry), manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter "CU") as a thermal sol-gel change agent (Sometimes referred to as “curdlan”) was prepared in the same manner as in Example 1 except that the addition amount shown in Table 2 was used.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
Instead of human mesenchymal stem cell PT-2501 (hMSC), human ES cell KhES-1 (Research Center for Regenerative Medicine, Kyoto University) (hereinafter sometimes referred to as “KhES-1”) is used. Cells were seeded in the same manner as in Example 1 except that the cell concentration at the time of seeding was the cell concentration shown in Table 2 and the temperature of the culture solution at the time of seeding was the temperature shown in Table 2. did.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 2.

As a result, as shown in Table 2, the cell growth rate was 9.6 times, the cell sedimentation rate was 85.0%, the cell death rate was 5.0%, and the score was 7.8.

<Example 11>
<Culture solution preparation process>
A culture solution was prepared in the same manner as in Example 10.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
After the culture solution preparation step, the culture solution injected into the culture vessel was heat-treated in the same manner as in Example 10.

<< Seeding process >>
After the temperature raising step, a seeding step was performed in the same manner as in Example 10.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 10.

<< Recovery process >>
After the culture step, a recovery step was performed in the same manner as in Example 10.

As a result, as shown in Table 2, the cell growth rate was 9.6 times, the cell sedimentation rate was 98.0%, the cell mortality rate was 4.0%, and the score was 9.0.

<Example 12>
<Culture solution preparation process>
A culture solution was prepared in the same manner as in Example 10.
The prepared culture solution was injected into the culture container in the amount shown in Table 2 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 2, the cell growth rate was 9.6 times, the cell sedimentation rate was 91.0%, the cell death rate was 12.0%, and the score was 7.7.

<Description of the results of Examples 10 to 12>
In Example 10, the cell sedimentation rate was lower than that in Example 11. This is considered to be because the temperature of the temperature lowering treatment was close to the solubilization temperature and the sol-gel conversion became unstable.
Example 12 had a higher cell mortality rate than Example 12. This is probably because the temperature of the temperature lowering process was low, and temperature stress was applied to the cells.

[Examples 13 to 19]
Examples 13 to 19 are examples showing the effect of the content of cellulose nanofibers.

<Example 13>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that cellulose nanofibers were not added and methyl cellulose (MC) was used as a thermal sol-gel change agent in an addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution injected into the culture vessel was subjected to heat treatment at the temperature described in the “temperature of heat treatment” column of “temperature raising step” in Table 3 for the time described in the “time of heat treatment” column.

<< Seeding process >>
Cells in which human embryonic stem cells H9 (WiCell Research Institute, Inc., Madison, Wis., USA) were used instead of human mesenchymal stem cells PT-2501 (hMSC), and the cell concentrations at the time of seeding are shown in Table 3. Cells were seeded in the same manner as in Example 1 except that the concentration and the temperature of the culture solution at the time of seeding were the temperatures shown in Table 3.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 3.

<< Culture process >>
After the sowing step, the culturing step was performed in the same manner as in Example 1 except that an incubator set to the temperature described in the column of “Average temperature of culture solution” in Table 3 was used.

<< Recovery process >>
After the culturing step, the recovery step was performed in the same manner as in Example 1 except that the temperature of the temperature lowering treatment was set to the temperature described in the column of “Temperature of the temperature lowering treatment” in Table 3 “Recovery step”. It was.

As a result, as shown in Table 3, the cell growth rate was 6.2 times, the cell sedimentation rate was 82.0%, the cell death rate was 5.0%, and the score was 4.8.

<Example 14>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
TEMPO-modified cellulose nanofiber (TEMPO1) was prepared in the same manner as in Comparative Example 1. The average diameter and carboxy group introduction amount of the prepared TEMPO 1 were measured in the same manner as in Comparative Example 1, and as shown in Table 2.

(Preparation of culture solution)
The culture solution was the same as in Example 1 except that the prepared TEMPO1 was used in the addition amount shown in Table 3 and that methylcellulose (MC) was used as the thermal sol-gel modifier in the addition amount shown in Table 3. Was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 13.

<< Seeding process >>
After the temperature raising step, cells were seeded in the same manner as in Example 13.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 13.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 13.

As a result, as shown in Table 3, the cell growth rate was 8.2 times, the cell sedimentation rate was 88.0%, the cell death rate was 5.0%, and the score was 6.9.

<Example 15>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 14 except that TEMPO1 was used in the addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 14.

As a result, as shown in Table 3, the cell growth rate was 8.5 times, the cell sedimentation rate was 93.0%, the cell death rate was 4.0%, and the score was 7.6.

<Example 16>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 14 except that TEMPO1 was used in the addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 14.

As a result, as shown in Table 3, the cell growth rate was 9.6 times, the cell sedimentation rate was 97.0%, the cell death rate was 5.0%, and the score was 8.8.

<Example 17>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 14 except that TEMPO1 was used in the addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 14.

As a result, as shown in Table 3, the cell growth rate was 9.2 times, the cell sedimentation rate was 91.0%, the cell mortality rate was 4.0%, and the score was 8.0.

<Example 18>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 14 except that TEMPO1 was used in the addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 14.

As a result, as shown in Table 3, the cell growth rate was 8.5 times, the cell sedimentation rate was 91.0%, the cell death rate was 5.0%, and the score was 7.3.

<Example 19>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 14 except that TEMPO1 was used in the addition amount shown in Table 3.
The prepared culture solution was injected into the culture container in the amount shown in Table 3 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 14.

As a result, as shown in Table 3, the cell growth rate was 5.2 times, the cell sedimentation rate was 90.0%, the cell mortality rate was 6.0%, and the score was 4.4.

<Description of the results of Examples 13 to 19>
As the CNF content increases, the gelation temperature Tc decreases.
Example 13, which does not contain CNF, has low cell growth rate and cell sedimentation. This is presumably because the gelation temperature Tc and the solation temperature Tcs are both high, and the temperature of the heat treatment in the temperature raising step and the temperature of the temperature lowering treatment in the recovery step are both high.
Example 19 has a low cell proliferation rate. This is probably because the gel strength was strong and the oxygen concentration inside the culture broth was reduced.

[Example 20 to Example 22]
Examples 20 to 22 are examples showing the effect of the type of CNF and the effect of the carboxyl group of CNF.

<Example 20>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
TEMPO-modified cellulose nanofibers (hereinafter “TEMPO3”) were prepared in the same manner as in Comparative Example 1 except that the amount of the oxidizing agent used and the pressure of the ultrahigh pressure homogenizer were adjusted so as to obtain the average diameter and carboxy group content shown in Table 4. Was prepared in some cases. The average diameter and carboxy group introduction amount of the prepared TEMPO 3 were measured in the same manner as in Comparative Example 1, and as shown in Table 4.

(Preparation of culture solution)
It replaces with TEMPO1 and is the same as Example 1 except the point which used the prepared TEMPO3 by the addition amount shown in Table 4, and the point which used methylcellulose (MC) by the addition amount shown in Table 4 as a thermal sol gel change agent. Thus, a culture solution was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution poured into the culture vessel was subjected to heat treatment at the temperature described in the “temperature treatment step C” column of the “temperature raising step” in Table 4 for the time described in the “time for heat treatment” column.

<< Seeding process >>
Instead of human mesenchymal stem cell PT-2501 (hMSC), human ES cell KhES-1 (Research Center for Regenerative Medicine, Kyoto University) (hereinafter sometimes referred to as “KhES-1”) is used. Cells were seeded in the same manner as in Example 1 except that the cell concentration at the time of seeding was the cell concentration shown in Table 4 and the temperature of the culture solution at the time of seeding was the temperature shown in Table 4. did.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 4.
<< Culture process >>
After the sowing step, the culturing step was performed in the same manner as in Example 1 except that an incubator set to the temperature described in the column of “Average temperature of culture broth” in Table 4 was used.

<< Recovery process >>
After the culturing step, the recovering step was performed in the same manner as in Example 1 except that the temperature of the temperature lowering treatment was set to the temperature described in the column of “Temperature lowering treatment temperature ° C.” of “Recovering step” in Table 4. It was.

As a result, as shown in Table 4, the cell growth rate was 9.6 times, the cell sedimentation rate was 98.0%, the cell death rate was 3.0%, and the score was 9.1.

<Example 21>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
Except that the amount of carboxymethylating agent used and the pressure of the high-pressure homogenizer were adjusted so as to achieve the average diameter and carboxy group content shown in Table 4, CM-modified cellulose nanofibers (hereinafter “ CM2 ”in some cases) was prepared. The average diameter of the prepared CM2 and the amount of carboxy group introduced were measured in the same manner as in Comparative Example 1, and as shown in Table 4.

(Preparation of culture solution)
A culture solution was prepared in the same manner as in Example 20 except that the prepared CM2 was used in the addition amount shown in Table 4 instead of TEMPO3.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
After the temperature raising step, cells were seeded in the same manner as in Example 20.

<< Culture process >>
After the sowing step, the culturing step was performed in the same manner as in Example 20.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 20.

As a result, as shown in Table 4, the cell growth rate was 9.0 times, the cell sedimentation rate was 93.0%, the cell death rate was 6.0%, and the score was 7.9.

<Example 22>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
In accordance with the method described in International Publication No. 2015/111734 ([0039]), the cellulose-based raw material is subjected to wet atomization treatment using a high-pressure homogenizer and fibrillated, whereby mechanically pulverized cellulose nanofibers (hereinafter referred to as “pulverized cellulose nanofiber”). “MEC1” may be referred to)). When the average diameter of the prepared MEC1 was measured in the same manner as in Comparative Example 1, it was as shown in Table 4. Since MEC1 is not subjected to carboxy group introduction treatment such as oxidation treatment, the carboxy group introduction amount is 0.0 mmol / g.

(Preparation of culture solution)
A culture solution was prepared in the same manner as in Example 20 except that the prepared MEC1 was used in the addition amount shown in Table 4 instead of TEMPO3.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

As a result, as shown in Table 4, the cell growth rate was 8.1 times, the cell sedimentation rate was 89.0%, the cell death rate was 9.0%, and the score was 6.6.

<Description of the results of Examples 20 to 22>
In both cell proliferation rate and cell death rate, Example 20 (TEMPO-modified CNF) was the best, and Example 21 (CM-converted CNF) was the next best.
In Example 22 (mechanical pulverization CNF), the gelation temperature Tc tends to be high, the heat treatment temperature is increased, the growth rate is slightly lowered, the solation temperature Tcs is slightly lowered, and the temperature lowering treatment temperature is lowered. It is thought that the cell mortality rate increased slightly.

[Example 23, Example 24]
Examples 23 and 24 are examples showing the effect of the type of thermal sol-gel change agent.

<Example 23>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
A TEMPO-modified cellulose nanofiber (hereinafter referred to as “TEMPO4”) was prepared in the same manner as in Comparative Example 1 except that the amount of the oxidizing agent used and the pressure of the ultrahigh pressure homogenizer were adjusted so as to obtain the average diameter and carboxy group content shown in Table 4. Was prepared in some cases. The average diameter and carboxy group introduction amount of the prepared TEMPO 4 were measured in the same manner as in Comparative Example 1, and as shown in Table 4.

(Preparation of culture solution)
It replaces with TEMPO1 and is the same as Example 1 except the point which used the prepared TEMPO4 by the addition amount shown in Table 4, and the point which used the methylcellulose (MC) by the addition amount shown in Table 4 as a thermal sol gel change agent. Thus, a culture solution was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
Cells in which human embryonic stem cells H9 (WiCell Research Institute, Inc., Madison, Wis., USA) are used in place of human mesenchymal stem cells PT-2501 (hMSC), and the cell concentrations at the time of seeding are shown in Table 4. Cells were seeded in the same manner as in Example 1 except that the concentration and the temperature of the culture solution at the time of seeding were the temperatures shown in Table 4.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 4.

<< Culture process >>
After the sowing step, the culturing step was performed in the same manner as in Example 1 except that an incubator set to the temperature described in the column of “Average temperature of culture broth” in Table 4 was used.

As a result, as shown in Table 4, the cell growth rate was 9.7 times, the cell sedimentation rate was 98.0%, the cell death rate was 3.0%, and the score was 9.2.

<Example 24>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 23 except that curdlan (CU) was used as the thermal sol-gel changing agent in the addition amount shown in Table 4.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
After the temperature raising step, cells were seeded in the same manner as in Example 23.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 23.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 23.

As a result, as shown in Table 4, the cell growth rate was 8.3 times, the cell sedimentation rate was 91.0%, the cell death rate was 7.0%, and the score was 7.0.

<Description of Results of Example 23 and Example 24>
Example 23 (MC) is superior to Example 24 (curdlan) in terms of cell growth rate and cell sedimentation, and the cell mortality rate is slightly smaller.

[Example 25, Comparative Example 2]
Example 25 and Comparative Example 2 are examples in which the temperature lowering operation in the recovery process is changed. Example 25 is an example in which cells were sedimented by a temperature lowering process, and Comparative Example 2 was an example in which cells were sedimented by a centrifuge process.

<Example 25>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
TEMPO-modified cellulose nanofibers (hereinafter “TEMPO5”) were prepared in the same manner as in Comparative Example 1 except that the amount of the oxidizing agent used and the pressure of the ultrahigh pressure homogenizer were adjusted so as to obtain the average diameter and carboxy group content shown in Table 4. Was prepared in some cases. The average diameter and carboxy group introduction amount of the prepared TEMPO 5 were measured in the same manner as in Comparative Example 1, and as shown in Table 4.

(Preparation of culture solution)
It replaces with TEMPO1 and is the same as Example 1 except the point which used the prepared TEMPO5 by the addition amount shown in Table 4, and the point which used methylcellulose (MC) as the thermal sol gel change agent by the addition amount shown in Table 4. Thus, a culture solution was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
Instead of human mesenchymal stem cell PT-2501 (hMSC), human ES cell KhES-1 (Research Center for Regenerative Medicine, Kyoto University) (hereinafter sometimes referred to as “KhES-1”) is used. Cells were seeded in the same manner as in Example 1 except that the cell concentration at the time of seeding was the cell concentration shown in Table 4 and the temperature of the culture solution at the time of seeding was the temperature shown in Table 4. did.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 4.

As a result, as shown in Table 4, the cell growth rate was 9.4 times, the cell sedimentation rate was 95.0%, the cell death rate was 5.0%, and the score was 8.5.

<Comparative example 2>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 25.
The prepared culture solution was injected into the culture container in the amount shown in Table 4 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
After the culture solution preparation step, the seeding step was performed in the same manner as in Example 25 without performing the temperature raising step.

<< Culture process >>
After the seeding process, the culture process was performed in the same manner as in Example 25.

<< Recovery process >>
After the culturing step, the temperature lowering operation was performed by centrifuge treatment (relative centrifugal acceleration 335 × g).

As a result, as shown in Table 4, the cell growth rate was 5.5 times, the cell sedimentation rate was 85.0%, the cell death rate was 51.0%, and the score was 2.3.

<Description of Results of Example 25 and Comparative Example 2>
Compared to Example 25, in which the cells were sedimented by the temperature lowering treatment, Comparative Example 2 in which the cells were sedimented by the centrifuge treatment had a clearly higher cell death rate.
This is considered to be due to the fact that the relative centrifugal acceleration during the centrifuge treatment was large and the cells were excessively stressed, resulting in an increase in cell mortality.

[Example 26, Comparative Example 3]
Example 26 and Comparative Example 3 are examples showing the effect of a floating material (gellan gum) other than CNF.

<Example 26>
<< Culture solution preparation process >>
(Preparation of cellulose nanofiber)
TEMPO-modified cellulose nanofibers (hereinafter “TEMPO6”) were prepared in the same manner as in Comparative Example 1 except that the amount of the oxidizing agent used and the pressure of the ultrahigh pressure homogenizer were adjusted so as to obtain the average diameter and carboxy group content shown in Table 5. Was prepared in some cases. The average diameter and carboxy group introduction amount of the prepared TEMPO 6 were measured in the same manner as in Comparative Example 1, and as shown in Table 5.

(Preparation of culture solution)
It replaces with TEMPO1 and is the same as Example 1 except the point which used the prepared TEMPO6 by the addition amount shown in Table 5, and the point which used methylcellulose (MC) as the thermal sol-gel change agent by the addition amount shown in Table 5. Thus, a culture solution was prepared.
The prepared culture solution was injected into the culture container in the amount shown in Table 5 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution poured into the culture vessel was subjected to heat treatment at the temperature described in the column “Heat treatment temperature ° C.” of Table 5 “Temperature raising step” for the time indicated in the “Heat treatment time” column.

<< Seeding process >>
Instead of human mesenchymal stem cell PT-2501 (hMSC), human ES cell KhES-1 (Research Center for Regenerative Medicine, Kyoto University) (hereinafter sometimes referred to as “KhES-1”) is used. Cells were seeded in the same manner as in Example 1 except that the cell concentration at the time of seeding was the cell concentration shown in Table 5 and the temperature of the culture solution at the time of seeding was the temperature shown in Table 5. did.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 5.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 1 except that an incubator set to the temperature described in the column of “Average temperature of culture solution” in Table 5 was used.

<< Recovery process >>
After the culturing step, the recovery step was performed in the same manner as in Example 1 except that the temperature lowering treatment temperature was set to the temperature described in the column “Temperature lowering treatment temperature ° C.” in Table 5 “Recovery step”. It was.

As a result, as shown in Table 5, the cell proliferation rate was 9.5 times, the cell sedimentation rate was 90.0%, the cell mortality rate was 4.0%, and the score was 8.2.

<Comparative Example 3>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 25 except that gellan gum (deacylated gellan gum KELCOGEL CG-LA, manufactured by Sanki Co., Ltd.) was used in the addition amount shown in Table 5 instead of TEMPO6. .
The prepared culture solution was injected into the culture container in the amount shown in Table 5 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
After the culture solution preparation step, the sowing step was performed in the same manner as in Example 26 without performing the temperature raising step.

<< Seeding process >>
After the temperature raising step, cells were seeded in the same manner as in Example 26.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 26.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 26.

As a result, as shown in Table 5, the cell growth rate was 8.0 times, the cell sedimentation rate was 35.0%, the cell death rate was 6.0%, and the score was 2.6.

<Description of Results of Example 26 and Comparative Example 3>
Comparative Example 3 (gellan gum) did not undergo sol-gel transfer, and cell sedimentation and cell recovery were inferior to Example 26 (CNF).

[Example 27, Example 28]
Example 27 and Example 28 are examples in which the seeding process is performed before the temperature raising process (Example 27) and the seeding process is performed after the temperature raising process (Example 28). is there.

<Example 27>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 1 except that methylcellulose (MC) was used as the thermal sol-gel changing agent in the addition amount shown in Table 5.
The prepared culture solution was injected into the culture container in the amount shown in Table 5 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

<< Seeding process >>
After the culture solution preparation step, the cells were seeded while keeping the temperature of the culture solution below Tc [° C.].
For cell seeding, human mesenchymal stem cells PT-2501 (hMSC) were used in the same manner as in Example 1, and the cell concentration at the time of seeding was set to the cell concentration shown in Table 5 and the culture solution at the time of seeding was used. Cells were seeded in the same manner as in Example 1 except that the temperature was set as shown in Table 5.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 5.

《Temperature raising process》
After the seeding process, a temperature raising process was performed.
The culture solution in the culture vessel was subjected to heat treatment at the temperature described in the “temperature of heat treatment” column of the “temperature raising step” in Table 5 for the time described in the “time of heat treatment” column.

As a result, as shown in Table 5, the cell growth rate was 9.3 times, the cell sedimentation rate was 93.0%, the cell death rate was 3.0%, and the score was 8.4.

<Example 28>
<< Culture solution preparation process >>
A culture solution was prepared in the same manner as in Example 27.
The prepared culture solution was injected into the culture container in the amount shown in Table 5 and used in the subsequent steps.
Further, the phase transition temperatures (Tc and Tcs) of the prepared culture were measured in the same manner as in Example 1.

《Temperature raising process》
A heating step was performed after the culture solution preparation step.
The culture solution in the culture vessel was subjected to heat treatment at the temperature described in the “temperature of heat treatment” column of the “temperature raising step” in Table 5 for the time described in the “time of heat treatment” column.

<< Seeding process >>
After the temperature raising step, the cells were seeded while maintaining the temperature of the culture solution at Tcs or higher.
As in Example 1, cell seeding was performed using hMSC, the cell concentration at the time of seeding was the cell concentration shown in Table 5, and the temperature of the culture solution at the time of seeding was the temperature shown in Table 5 Cells were seeded in the same manner as in Example 1 except for.
The state of the culture solution at the time of seeding and the temperature of the culture solution were as shown in the corresponding column of “Seeding step” in Table 5.

<< Culture process >>
After the seeding step, the culture step was performed in the same manner as in Example 27.

<< Recovery process >>
After the culturing step, a collecting step was performed in the same manner as in Example 27.

<Description of the results of Example 27 and Example 28>
There was no difference in the effect between Example 27 and Example 28.

Claims

A temperature raising step for raising the temperature of the culture medium having a temperature lowering phase transition temperature Tcs, which is lower than the temperature rising temperature transition temperature Tc, from a temperature lower than Tc to a temperature higher than Tc;
In the temperature raising step, after the temperature of the culture solution is raised to Tc or higher, a culture step in which cells are suspended in the culture solution at a culture temperature of Tcs or higher,
A method for culturing cells.
Here, the temperature transition phase transition temperature Tc is the temperature of the culture solution when the culture solution is heated from 3.0 ° C. to 98.0 ° C. at a temperature increase rate of 5.0 ° C./min. The temperature at which the viscosity of the culture solution becomes 10 × ηs when the viscosity at 3.0 ° C. is ηs, and the temperature transition phase transition temperature Tcs during cooling is 5.0 ° C./min for the culture solution. In the case where the temperature is lowered from 98.0 ° C. to 3.0 ° C. at the rate of temperature decrease, this is the temperature at which the viscosity of the culture solution becomes 10 × ηs, the temperature unit is ° C., and the viscosity unit is Pa · s. And
The method for culturing cells according to claim 1, wherein Tc and Tcs satisfy the following formula (1).
1.0 ° C ≦ Tc−Tcs ≦ 70.0 ° C (1)
After the culturing step,
A recovering step of lowering the temperature of the culture solution to less than Tcs, and precipitating and recovering the cells cultured in the culture step;
The cell culturing method according to claim 1, comprising:
The method for culturing cells according to any one of claims 1 to 3, wherein the temperature transition phase transition temperature Tcs during cooling is from 3.0 ° C to 41.0 ° C.
The method for culturing cells according to any one of claims 1 to 4, wherein the culture solution contains cellulose nanofibers having an average diameter of 2.0 nm to 100 nm and a thermal sol-gel changing agent.
The method for culturing cells according to claim 5, wherein the content of carboxy groups in the cellulose nanofiber is 0.60 mmol / g or more and 2.0 mmol / g or less.
The method for culturing cells according to claim 5 or 6, wherein the content of the cellulose nanofiber is 0.01% by mass or more and 1.0% by mass or less in the culture solution.
The cell culture method according to any one of claims 5 to 7, wherein the cellulose nanofibers are oxidized.
The method for culturing cells according to any one of claims 5 to 8, wherein the content of the thermal sol-gel changing agent is 0.05% by mass or more and 3.0% by mass or less in the culture solution.
The cell culture method according to any one of claims 5 to 9, wherein the thermal sol-gel changing agent is methylcellulose.
The method for culturing cells according to any one of claims 1 to 10, wherein the cells are stem cells.
After the temperature raising step and before the culturing step,
The cell culturing method according to any one of claims 1 to 11, further comprising a post-gelation seeding step of seeding cells in the culture solution after the culture solution is heated to Tc or higher in the temperature raising step. .
Before the temperature raising step,
The cell culturing method according to any one of claims 1 to 11, further comprising a pre-gelation seeding step of seeding the culture medium with cells.
A culture solution having a temperature transition phase transition temperature Tc and a temperature transition phase transition temperature Tcs.
Here, the temperature transition phase transition temperature Tc is the temperature of the culture solution when the culture solution is heated from 3.0 ° C. to 98.0 ° C. at a temperature increase rate of 5.0 ° C./min. The temperature at which the viscosity of the culture solution becomes 10 × ηs when the viscosity at 3.0 ° C. is ηs, and the temperature transition phase transition temperature Tcs during cooling is 5.0 ° C./min for the culture solution. In the case where the temperature is lowered from 98.0 ° C. to 3.0 ° C. at the rate of temperature decrease, this is the temperature at which the viscosity of the culture solution becomes 10 × ηs, the temperature unit is ° C., and the viscosity unit is Pa · s. And
The culture solution according to claim 14, wherein Tc and Tcs satisfy the following formula (1).
1.0 ° C ≦ Tc−Tcs ≦ 70.0 ° C (1)
The culture solution according to claim 14 or 15, wherein the temperature drop phase transition temperature Tcs is 3.0 ° C or higher and 41.0 ° C or lower.
The culture solution according to any one of claims 14 to 16, comprising cellulose nanofibers having an average diameter of 2.0 nm to 100 nm and a thermal sol-gel changing agent.
The culture solution according to claim 17, wherein the carboxy group content of the cellulose nanofiber is 0.60 mmol / g or more and 2.0 mmol / g or less.
The cell culture solution according to claim 17 or 18, comprising the cellulose nanofibers in an amount of 0.01% by mass to 1.0% by mass.
The culture solution according to any one of claims 17 to 19, wherein the cellulose nanofibers are oxidized.
The culture solution according to any one of claims 17 to 20, comprising 0.05% by mass to 3.0% by mass of the thermal sol-gel change agent.
The culture solution according to any one of claims 17 to 21, wherein the thermal sol-gel changing agent is methylcellulose.
The culture solution according to any one of claims 14 to 22, for use in the cell culture method according to any one of claims 1 to 13.