Additives to suspension culture medium, medium composition, and culture method

Abstract

Cells are cultured in a suspended state, and the cultured cells can be easily recovered. The additive for suspension culture according to the embodiment is a medium additive added to the medium to culture cells in a suspended state, and includes a cellulose oligomer. The medium composition according to the embodiment is a medium composition capable of culturing cells in a suspended state, and includes a cellulose oligomer. The method of culturing cells according to the embodiment includes culturing cells in this medium composition.

Description

Technology Field

[0001] The present invention relates to a suspension culture medium additive used for suspending and culturing cells, a medium composition capable of suspending and culturing cells, and a method of culture using the same. Background Technology

[0002] Three-dimensional cell culture is known as a method of culturing cells while allowing them to interact three-dimensionally with their surrounding environment. In three-dimensional cell culture, for example, if cells are cultured in a liquid medium, the cells will settle and aggregate. To prevent cells from aggregating, there are methods such as using hydrogels or suspending the cells by stirring with a spinner.

[0003] However, in the method using hydrogels, there are problems such as the growth of cells accepted in the gel matrix being hindered by the pressure of the gel, the cell aggregates being prone to becoming non-uniform in size due to the non-uniform structure of the gel, and it being difficult to separate the gel from the cells. In addition, in agitated culture using a spinner, there is a concern that the cells may be damaged by constant agitation.

[0004] Patent Document 1 discloses a suspension culture method in which nanofibers, such as cellulose or chitin, are added to a liquid medium to culture cells, as a method for culturing cells by suspending them without stirring with a spinner. However, the cellulose nanofibers disclosed in Patent Document 1 are polymer compounds obtained by micronizing (grinding) cellulose using a high-pressure homogenizer or the like, and generally, the aqueous dispersion thereof has high viscosity, making it difficult to recover the cultured cells. Prior art literature

[0005] International Publication WO2015 / 111686 The problem to be solved

[0006] The embodiments of the present invention aim to provide a suspension culture medium additive, a medium composition, and a culture method that allow cells to be cultured in a suspended state and the cultured cells to be easily recovered. means of solving the problem

[0007] The additive for suspension culture according to an embodiment of the present invention is a medium additive added to a medium to suspend and culture cells, and comprises a cellulose oligomer.

[0008] A culture medium composition according to an embodiment of the present invention is a culture medium composition capable of suspending and culturing cells, and comprises a cellulose oligomer.

[0009] In the present invention, the cellulose oligomer may not have a substituent, or it may have a substituent.

[0010] A cell culture method according to an embodiment of the present invention comprises culturing cells in this medium composition. Effects of the invention

[0011] In an embodiment of the present invention, by incorporating a cellulose oligomer into the culture medium, cells can be cultured in a suspended state. Furthermore, since the culture medium maintains a low viscosity even after the addition of the cellulose oligomer, cells can be easily recovered, for example, by filtration. Brief explanation of the drawing

[0012] Figure 1 is a photograph of a dispersion showing the results of evaluating the dispersion stability of cellulose oligomers in ultrapure water and DMEM. Figure 2 is a graph showing the results of DLS measurements of cellulose oligomers. Figure 3 is a microscopic image of the culture medium after 1 day of suspension culture. Figure 4 shows microscopic images of the culture medium after 1 day of suspension culture, (A) before medium exchange and (B) after medium exchange. Figure 5 (A) is a microscopic image of the culture medium after suspension culture for 5 days using a cellobiose-derived oligomer, and (B) is a glucose-derived cellulose oligomer. Figure 6 shows microscopic images of (A) before culture and (B) after culture, taken by fixing the observation point of cells cultured for 2 days without changing the medium. Figure 7 (A) is a microscopic image of a spheroid cultured and recovered using a cellobiose-derived oligomer, and (B) is a spheroid cultured and recovered using a glucose-derived cellulose oligomer, respectively. Specific details for implementing the invention

[0013] 1. Additives to suspension culture media

[0014] The medium additive for suspension culture according to the present embodiment (hereinafter simply referred to as the medium additive) comprises a cellulose oligomer. Since the cellulose oligomer is water-insoluble and has excellent dispersion stability in water, it has the effect of maintaining cells in a suspended state in a liquid medium. Therefore, it can be used as an additive for culturing cells in a suspended state.

[0015] (A) Cellulose oligomer

[0016] Cellulose oligomers are oligosaccharides having a structure in which glucose is linked by β-1,4 glycosidic bonds, and are also called celluloligosaccharides. Cellulose oligomers may not have substituents, or they may have substituents such as alkyl groups on the anomer at the reducing end, for example.

[0017] It is preferable that the cellulose oligomer has an average degree of polymerization (DP) (the average value of the number of glucose units present in one molecule) of 5 to 20. It is preferable that the average degree of polymerization (DP) be 6 or higher, and also preferable that it be 15 or lower, more preferably 12 or lower, and may be 10 or lower. The cellulose oligomer is typically a mixture of compounds with different degrees of polymerization, for example, may include those with a degree of polymerization of 4 to 20, may include those with a degree of polymerization of 5 to 18, and may include those with a degree of polymerization of 5 to 13.

[0018] As a cellulose oligomer, it is preferable to use a compound represented by the following general formula (1).

[0019]

[0020] In the formula, A represents a hydrogen atom or a substituent. The substituent is a group introduced in place of a hydrogen atom, and examples include an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 5 carbon atoms. Additionally, the dashed line in the bond between the carbon at the first position of the reduction end and the OA group indicates that the OA group may be of the α type or the β type. n represents the average degree of polymerization and is preferably 5 to 20. n is preferably 6 or higher, more preferably 15 or lower, more preferably 12 or lower, and may be 10 or lower.

[0021] The method of synthesizing cellulose oligomers is not particularly limited. For example, a method may be used in which α-glucose-1-phosphate (hereinafter referred to as αG1P) and at least one primer selected from the group consisting of glucose, cellobiose, and their derivatives are reacted with cellodextrin phosphorylase (hereinafter referred to as CDP). This reaction is a synthesis method utilizing the reverse reaction of CDP, wherein αG1P is used as a glucose donor and at least one selected from the group consisting of glucose, cellobiose, and their derivatives is used as a primer (i.e., glucose acceptor), and by reacting these with CDP, αG1P is gradually polymerized as a monomer with respect to the primer.

[0022] For example, the reaction when the primer is glucose is as shown below.

[0023]

[0024] Also, the reaction when the primer is cellobiose is as shown below.

[0025]

[0026] In addition, by replacing the hydrogen atoms of the hydroxyl groups on the anomer in the primer with substituents such as alkyl groups, it is possible to synthesize cellulose oligomers having various substituents represented by formula (1).

[0027] It is known that CDP is produced by microorganisms such as Clostridium thermocellum and Cellulomonas, and can be obtained by known methods using these microorganisms. For example, CDP derived from Clostridium thermocellum YM4 can be prepared by an E. coli expression system in accordance with the method described in M. Krishnareddy et al., J. Appl. Glycosci., 2002, 49, 1-8, but is not limited to this.

[0028] The concentration of CDP is not specifically limited and, for example, may be 0.1 U / ml or higher, or 0.2 U / ml or higher. Here, the amount of CDP enzyme can be determined, for example, based on enzyme activity. In this case, for example, αG1P, D-(+)-cellobiose, and CDP are incubated, and the phosphate produced by CDP is quantified, so that the amount of enzyme releasing 1 μmol of phosphate per minute can be set to 1 U.

[0029] For example, 10 to 1000 mM of αG1P, 10 to 200 mM of primer (glucose, cellobiose and their derivatives), and 0.1 U / mL or more of CDP are mixed in 100 to 1000 mM of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.0 to 8.0), and the mixture is reacted by incubating at 10 to 80°C for 30 minutes to 30 days to synthesize the cellulose oligomer of formula (1).

[0030] The cellulose oligomer synthesized in this way has a cellulose type II crystal structure. While naturally derived cellulose has a cellulose type I crystal structure in which cellulose chains are arranged in parallel, the artificially synthesized cellulose oligomer forms a thermodynamically stable cellulose type II crystal structure.

[0031] As for the cellulose oligomer, the one synthesized using CDP as described above may be used as is, or a self-organized one may be used. Self-organized cellulose oligomers can be prepared as follows. Specifically, an aqueous solution is prepared by dissolving the cellulose oligomer in an alkaline aqueous solution, and the resulting aqueous solution is adjusted to neutral or acidic using an acid. This causes the cellulose oligomer to become insoluble and precipitate; at that time, the cellulose oligomer self-organizes by incubating it while stirring, vibrating, or in a standing state. That is, the molecules of the cellulose oligomer align to form a cellulose structure having a cellulose type II crystal structure.

[0032] (B) Medium additive

[0033] The medium additive may be an aqueous dispersion of cellulose oligomers. That is, it is preferable that the medium additive contains water along with cellulose oligomers, and disperses the cellulose oligomers in this water. Since the medium additive is an aqueous dispersion, the cellulose oligomers can be easily dispersed in the liquid medium. Additionally, the medium additive may consist solely of cellulose oligomers.

[0034] In the case of the above aqueous dispersion, the concentration of the cellulose oligomer is not particularly limited, but, for example, it may be 0.01 to 2% (w / v) or 0.5 to 1% (w / v). Also, in this specification, “% (w / v)” means the mass (g) of the target component contained in 100 mL of solution.

[0035] The particle size of the cellulose oligomer in the aqueous dispersion of the cellulose oligomer is not particularly limited, but it is preferable that the particle diameter of the maximum intensity measured by dynamic light scattering (DLS) be 10 μm or less, more preferably 5 μm or less, and may be 1.0 μm or less. The lower limit is not particularly limited and may be 1 nm or more, 10 nm or more, or 100 nm or more.

[0036] Sodium chloride may be included in the aqueous dispersion of the cellulose oligomer. That is, the aqueous dispersion of the cellulose oligomer may be obtained by dispersing the cellulose oligomer in saline solution, and more specifically, by dispersing the cellulose oligomer in physiological saline solution.

[0037] In addition to the above components, the culture medium additive may include additives such as methylated cellulose, for example.

[0038] 2. Culture medium composition

[0039] The culture medium composition according to the present embodiment is a culture medium composition capable of suspending and culturing cells, and is characterized by including a cellulose oligomer. As the cellulose oligomer, the one described in 1 above may be used. That is, the culture medium composition according to the embodiment is formed by adding the above-mentioned suspension culture medium additive to the culture medium and mixing them.

[0040] As mentioned above, since cellulose oligomers have the effect of keeping cells suspended in a liquid medium, a medium composition containing cellulose oligomers is a liquid medium composition capable of culturing cells while they are suspended. Furthermore, cellulose oligomers are dispersed in a suspended state within the medium and keep cells suspended in that state; they do not solidify like hydrogels to retain and keep cells within the matrix. Therefore, it is believed that cellulose oligomers do not restrict the movement or growth of cells (including spheroids, which are cell aggregates), and thus, cells with a more uniform size and high roundness can be obtained.

[0041] The medium included in the medium composition can be appropriately selected depending on the type of cell, and various liquid media can be used. Specifically, examples include Dulbecco's modified Eagle medium (DMEM), Eagle's Minimum Essential Medium (EMEM), Minimum Essential Medium Eagle (Alpha Modification), Glasgow Minimum Essential Medium (GMEM), Nutrient Mixture F-12 Ham, Dulbecco's modified Eagle Medium / Nutrient Mixture F-12 Ham, Iscove's Modified Dulbecco's Medium (IMDM), RPMI-1640 medium, McCoy's 5A medium, etc.

[0042] The components included in the culture medium are not particularly limited, and various components generally used in culture media, such as amino acids, inorganic salts, glucose, and vitamins, can be included.

[0043] The concentration of cellulose oligomer in the culture medium composition is not particularly limited, but it is preferably 0.01 to 2% (w / v), and more preferably 0.5 to 1% (w / v). By having a concentration of cellulose oligomer of 0.01% (w / v) or higher, the dispersion stability of the cellulose oligomer in the culture medium composition is improved, allowing the cells to be suspended, and by having a concentration of 0.5% (w / v) or higher, the effect can be further enhanced. Also, by having a concentration of 2% (w / v) or lower, it can be easily handled as an aqueous dispersion when dispensing with a pipette, etc. Furthermore, when the concentration of cellulose oligomer is low, it may separate into two layers—a clear supernatant (upper layer) and a lower layer where the cellulose oligomer is dispersed—in a relatively short period of time; however, even in that case, since suspension culture can be performed in the lower layer where the cellulose oligomer is dispersed, suspension culture may be performed in such a separated state.

[0044] The method for preparing the culture medium composition is not particularly limited. For example, when using the aqueous dispersion of the cellulose oligomer, the aqueous dispersion and the liquid medium may be sterilized separately beforehand, and then the two may be mixed to uniformly disperse the cellulose oligomer in the culture medium composition. Alternatively, cells may be added and dispersed in the medium and then mixed with the aqueous dispersion of the cellulose oligomer, in which case the cells are also included in the culture medium composition at the stage where the culture medium composition is obtained. Or, the culture medium composition may be prepared by mixing the aqueous dispersion of the cellulose oligomer and the liquid medium to uniformly disperse the cellulose oligomer in the culture medium composition and then sterilizing. Furthermore, the sterilization of the aqueous dispersion of the cellulose oligomer may be considered as a sterilization treatment by preparing the aqueous dispersion via an alkaline aqueous solution, for example, as described in the examples below.

[0045] In the present embodiment, the cells are not particularly limited and may be of animal origin or of plant origin. Examples of cells of animal origin include germ cells, somatic cells, hepatocytes, progenitor cells, immortalized cells (cell lines), and genetically modified cells. Cells collected or isolated from living organisms and, in some cases, subjected to additional artificial manipulation may be used. Hepatocytes are cells that possess both self-proliferative and differentiation capabilities; examples include embryonic hepatocytes (ES cells), somatic hepatocytes (e.g., neural hepatocytes, hematopoietic hepatocytes, cutaneous hepatocytes, etc.), and induced pluripotent stem cells (iPS cells). Progenitor cells are cells that are in the intermediate stage of differentiation from hepatocytes into specific somatic cells or germ cells. Cell lines are cells that have acquired infinite proliferative capacity through artificial manipulation in vitro; examples include HeLa (a cell line derived from human cervical cancer).

[0046] The culture medium composition according to the present embodiment is capable of culturing cells by suspending them, that is, enabling suspension culture. Here, suspension culture refers to culturing cells while they are suspended in a liquid medium without adhering to a culture vessel. In the present embodiment, it is desirable that cells can be cultured by suspending them in the liquid culture medium composition without external pressure or vibration or stirring within the culture medium composition, that is, even when the culture medium composition is in a stationary state; therefore, it is desirable that the culture medium composition enables stationary suspension culture. The period during which cells are suspended in a stationary state, i.e., stationary suspension, is preferably 1 hour or longer, more preferably 24 hours or longer, and even more preferably 48 hours or longer.

[0047] The viscosity of the culture medium composition is not particularly limited, but it is preferable that the viscosity at 25°C be 5.00 mPa·s or less, and may be 3.00 mPa·s or less. Since it is preferable for the viscosity of the culture medium composition to be lower, the lower limit is not particularly limited and may be, for example, 0.50 mPa·s or more.

[0048] 3. Culture Method

[0049] The cell culture method according to the present embodiment includes culturing cells in the medium composition. Specifically, the cells are mixed so as to be uniformly dispersed in the medium composition, and the resulting culture solution is cultured in a culture vessel. The culture vessel is not particularly limited and may include, for example, a flask, a dish, a Petri dish, a multi-well plate, etc.

[0050] As a method for dispersing cells in a culture medium composition, a culture medium composition containing a cellulose oligomer may be prepared, and then cells may be added to and mixed in the culture medium composition, or cells may be added to and mixed in the culture medium, and then a cellulose oligomer may be added and mixed therein.

[0051] Culture may be performed in a static state, or the culture medium may be rotated, shaken, or stirred as necessary. Preferably, it is performed in a static state to reduce damage to the cells. In this embodiment, due to the cellulose oligomers dispersed in the culture medium, the cells are not concentrated only on the bottom surface of the culture medium but are spread and dispersed three-dimensionally, making static suspension culture possible.

[0052] In one embodiment, the cells may form a spheroid, which is a three-dimensional cell aggregate (a spherical cell polymer in which cells are aggregated and clustered together), by culture. The size of the spheroid is not particularly limited as it varies depending on the cell type and the culture period; for example, the diameter may be 20 to 1000 μm, 40 μm to 500 μm, or 50 to 300 μm.

[0053] Conditions such as temperature and time during culture can be appropriately set depending on the cells being cultured. For example, for animal cells, the culture temperature may typically be 25 to 39°C, preferably 33 to 39°C. The CO2 concentration may typically be 4 to 10 volume% in the culture atmosphere, preferably 4 to 6 volume%. The culture time is typically 3 to 35 days, but can be appropriately set according to the purpose of the culture. Also, for plant cells, the culture temperature may typically be 20 to 30°C, and if light is required, it may be conducted under illuminance conditions of 2,000 to 8,000 lux. Also, the culture time is typically 3 to 70 days, but can be appropriately set according to the purpose of the culture.

[0054] The cell concentration can also be appropriately set depending on the cells being cultured and is not particularly limited; for example, 1.0 × 10⁻⁶ as the cell concentration at the time of seeding (i.e., the step of seeding cells into the medium composition). 3 Pieces / mL~1.0×10⁻⁶ 10 It can be particles / mL, or 1.0×10 4 Pieces / mL~1.0×10⁻⁶ 8 It can be one / mL.

[0055] In the culture process, the medium may be exchanged. That is, the cell culture method according to the present embodiment may include a process of culturing cells in the medium composition, and a process of separating the cells obtained by this culture from the medium together with the cellulose oligomer, and mixing the separated cells and cellulose oligomer with a new medium to perform further culture. As a method for separating the cells from the medium together with the cellulose oligomer, for example, the cells and cellulose oligomer may be precipitated by centrifugation and the medium as the supernatant removed. By adding a new medium to the separated cells and cellulose oligomer and mixing them, the cells and cellulose oligomer can be redispersed in the culture medium. Therefore, suspension culture is possible without adding new cellulose oligomer, and the cellulose oligomer can be used repeatedly.

[0056] The culture method according to the present embodiment may also include a process for recovering cultured cells. Recovery is a process of separating cultured cells from the culture medium, and can be performed, for example, by filtration. The cellulose oligomer is dispersed in the culture medium, thereby maintaining the cells in a three-dimensionally dispersed state within the culture medium, and the cellulose oligomer and the cells are not bound to each other. Therefore, separation of the cellulose oligomer and the cells is easy. Furthermore, the cellulose oligomer does not substantially increase the viscosity of the aqueous dispersion, and thus the medium composition and the culture medium also have low viscosity. Consequently, filtration by natural filtration is possible, and damage to the cells can be reduced.

[0057] In one embodiment, the cultured cell (e.g., spheroid) is larger than the cellulose oligomer. For example, it is preferable to carry out the culture until the cell becomes larger than the cellulose oligomer. As the cellulose oligomer is smaller than the cultured cell (spheroid), the cultured cell can be easily separated and recovered from the medium and the cellulose oligomer by filtering using a filter that has a pore size larger than the particle size of the cellulose oligomer and smaller than the pore size of the cultured cell.

[0058] (Example)

[0059] The present invention is further explained below by way of examples, but is not limited to these.

[0060] 1. Reagents

[0061] α-glucose-1-phosphate (αG1P) disodium hydrate, DMEM, penicillin-streptomycin, and dulbeco phosphate buffer (DPBS) were purchased from Fujifilm Wako Junyaku Kogyo. Heavy water was purchased from Sigma-Aldrich. 40 mass% sodium deuterium heavy water solution was purchased from Cambridge Isotopes Laboratories. Ultrapure water was supplied by Milli-Q Systems (Milli-Q Advantage A-10, Merck Millipore). Other than that, special grade or higher reagents were purchased and used from Nakaraitesk.

[0062] 2. Experimental Method

[0063] (1) Synthesis of cellulose oligomer

[0064] A cellulose oligomer (derived from glucose) with an average degree of polymerization of 10 was synthesized according to the method described in T. Serizawa et al., Polym. J., 2016, 48, 539-544 (using D-glucose as a primer). In addition, a cellulose oligomer (derived from cellobiose) with an average degree of polymerization of 7 was synthesized according to the method described in T. Serizawa et al., Langmuir, 2017, 33, 13415-13422 (using cellobiose as a primer).

[0065] Specifically, 200 mM αG1P, 50 mM D-glucose or cellobiose, and 0.2 U / mL cellodextrin phosphorylase (CDP) were mixed in 500 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer (pH 7.5) and incubated at 60°C for 3 days. The reaction mixture containing the product was centrifuged (15,000 rpm, for at least 10 minutes, at 4°C), and after removing the supernatant, ultrapure water was added to redisperse the product. The operation of centrifuging (under the same conditions) was repeated until the substitution rate of the supernatant was 99.999% or higher, thereby obtaining a cellulose oligomer.

[0066] The average degree of polymerization of the product was measured using a proton nuclear magnetic resonance (NMR) device. The sample was prepared by dissolving at least 12 mg of freeze-dried product in 600 μL of a 4 mass% sodium deuterium heavy water solution. An ADVANCE III HD500 (Bruker Biospin, magnetic field strength: 500 MHz, 16 integration cycles) was used as the NMR device. The average degree of polymerization was calculated based on the integrated values ​​of protons at the anomeric groups (δ~5.1, 4.5) of the reducing end of the cellulose oligomer and other anomeric groups (δ~4.3).

[0067] (2) Preparation of an aqueous dispersion of cellulose oligomer

[0068] A predetermined amount of freeze-dried cellulose oligomer was dispersed by adding a 1N sodium hydroxide aqueous solution to a 1.5 mL tube. This was incubated at -20°C for 20 minutes and then dissolved by returning to room temperature to prepare a cellulose / sodium hydroxide aqueous solution. 0.18N hydrochloric acid containing phosphate buffer (pH 7.4) and the cellulose / sodium hydroxide aqueous solution were mixed such that the final concentration of sodium chloride was 137 mM and the final concentration of cellulose oligomer was 1% (w / v) and 2% (w / v) for cases using glucose-derived and cellobiose-derived cellulose oligomers, respectively, and left to stand at 25°C for 1 day. The reaction mixture containing the product was centrifuged (15,000 rpm, for at least 10 minutes, at 25°C), the supernatant was removed, sterile purified water was added to redisperse the product, and centrifuged (under the same conditions). By repeating this operation, the solution was purified until the substitution rate of the supernatant was 99.999% or higher, and an aqueous dispersion of cellulose oligomer was obtained.

[0069] (3) Evaluation of dispersion stability of cellulose oligomer

[0070] The aqueous dispersion of the cellulose oligomer prepared in (2) was adjusted to 1% (w / v), and 250 μL of the obtained aqueous dispersion was mixed with 250 μL of ultrapure water or 2×DMEM (DMEM at 2x concentration) in a 1 mL vial. The mixture was incubated at 37°C for a specified time, and the dispersibility of the cellulose oligomer was observed visually over time. Additionally, the dispersion incubated for 3 days was transferred to a 1.5 mL tube and centrifuged (15,000 rpm, 5 minutes, 25°C), and then 500 μL of ultrapure water or DMEM was added to redisperse the mixture in a 1 mL vial. This was incubated at 37°C for a specified time, and the dispersibility was observed in the same way.

[0071] (4) DLS measurement of cellulose oligomer dispersion

[0072] The aqueous dispersion of the cellulose oligomer prepared in (2) was adjusted to 0.005% (w / v), and 80 μL of the obtained aqueous dispersion was added to the cell and measured after standing at 25°C for 3 minutes. A Zetasizer Nano ZSP (Malvern) was used for the measurement, and the hydrodynamic diameter was calculated by cumulant analysis using polystyrene latex as a standard sample, with the temperature set to 25°C and the scattering angle to 173°. DLS measurements were performed 3 times (n=3), and the average value was obtained.

[0073] (5) Measurement of viscosity of the medium composition

[0074] (2) The cellulose oligomer aqueous dispersion prepared in (2) was adjusted to 1% (w / v), and 1 mL of the obtained aqueous dispersion and 1 mL of 2×DMEM were mixed in a 2 mL tube. Also, 1 mL of 0.2% (w / v) cellulose nanofiber aqueous dispersion (Leocrista, Daiichi Kokyose Iyaku) and 1 mL of 2×DMEM were mixed in a 2 mL tube. For the culture medium composition prepared in this way, a tuning fork viscometer (SV-1A, A&D, 30 Hz) was used, and the viscosity was measured at a measurement temperature of 25°C.

[0075] (6) Cell culture

[0076] HeLa cells (JCRB cell bank) were cultured in 10 cm dishes in DMEM containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2, and subcultured until approximately 80% confluence was reached. Subculture was performed by the following procedure: the medium was removed, 10 mL of DPBS was added to the dish and gently stirred. The DPBS was removed, 1 mL of 0.5 mg / mL trypsin solution was added and spread over the entire surface, and incubated for 5 minutes at 37°C and 5% CO2. 9 mL of DMEM was added to suspend the cells, 0.3 to 1 mL was transferred to a new dish, DMEM was added to make the total volume 10 mL, and the mixture was gently stirred and cultured at 37°C and 5% CO2.

[0077] (7) Suspension culture using cellulose oligomer dispersion

[0078] In (6), the cell suspension that was not used during subculture was transferred to a 15 mL tube and centrifuged (500 rpm, 3 minutes). After removing the supernatant, 10 mL of 2×DMEM was added to suspend the cells, and the centrifugation (under the same conditions) operation was repeated 3 times. A solution of the cell suspension mixed with 0.4% trypan blue solution in a 1:1 (volume ratio) was applied to a hemocytometer to count the number of live cells, and the cell concentration was 1.0×10 5 The solution was diluted with 2×DMEM to a volume of cells / mL. 50 μL of cell suspension and 50 μL of cellulose oligomer aqueous dispersion were mixed in a 96-hole plate and incubated for a specified time under conditions of 37°C and 5% CO2. As for the cellulose oligomer aqueous dispersion, the solution prepared in (2) was adjusted to 1.0% (w / v) and used. The medium was changed every day by the following procedure: the cell culture medium was transferred to a 1.5 mL tube, 1 mL of DMEM was added, and the mixture was centrifuged (200 g, 5 min, 25°C). After removing the supernatant, DMEM was added to redisperse the solution to a total volume of 100 μL, and the mixture was transferred to a 96-hole plate. The morphology of the cells was observed using a fluorescence microscope (ZOE fluorescence cell imager, Bio-rad) in bright-field mode. When fixing the observation point, a mark was made on the bottom surface of the 96-hole plate, and the height of the microscope was fixed for observation.

[0079] (8) Recovery of cultured cells

[0080] After suspending the cells for 5 days according to (7), the cell culture medium was transferred to a 1.5 mL tube and dispersed by adding 1 mL of DPBS. The dispersion was filtered using a nylon mesh filter (pore diameter 40 μm, Funakoshi). Cells remaining on the filter were recovered into a 96-hole plate by flowing 200 μL of DPBS from the opposite side of the filter, and their morphology was observed using a bright-field mode of a fluorescence microscope (ZOE fluorescence cell imager, Bio-rad).

[0081] 3. Results and Discussion

[0082] (1) Results of the dispersion stability evaluation of cellulose oligomer

[0083] Figure 1 shows the appearance of cellulose oligomers dispersed in ultrapure water and serum medium (DMEM) and left to stand for a set period of time. Although slight sedimentation was observed after standing for 3 days, all cellulose oligomers used were well dispersed in ultrapure water. Furthermore, the cellulose oligomers were well dispersed in serum medium, indicating that they can be stably dispersed even in solutions containing various substances such as proteins and inorganic salts. Additionally, when the cellulose oligomer dispersion that had been left to stand for 3 days was centrifuged, redispersed, and incubated again, the cellulose oligomers were well dispersed, and it was found that the dispersibility of the cellulose oligomers was maintained even after centrifugation and redispersion.

[0084] In FIG. 1, "Cel-derived (DMEM)" means a cellulose oligomer derived from Cellobiose dispersed in a serum medium, and "Cel-derived (MilliQ)" means a cellulose oligomer derived from Cellobiose dispersed in ultrapure water. "Glc-derived (DMEM)" means a cellulose oligomer derived from glucose dispersed in a serum medium, and "Glc-derived (MilliQ)" means a cellulose oligomer derived from glucose dispersed in ultrapure water.

[0085] (2) DLS measurement results of cellulose oligomer

[0086] Figure 2 shows the results of DLS measurements of a cellulose oligomer dispersion (derived from Cellobiose). Two peaks with particle diameters of 602 nm as the main component and 5719 nm as the minor component were observed. The particle diameter with maximum intensity was 602 nm. It was found that the cellulose aggregates generated by self-organization through neutralization had particle diameters ranging from sub-μm to several μm.

[0087] (3) Viscosity measurement results of the medium composition

[0088] The viscosity of a solution in which cellulose oligomers were dispersed in DMEM was measured. For the medium composition containing glucose-derived and cellobiose-derived cellulose oligomers at a concentration of 0.5% (w / v) used for suspension culture, the viscosities were 1.64±0.2 and 2.82±0.06 mPa·s, respectively. A medium composition containing 0.1% (w / v) of cellulose nanofibers, which are naturally derived and higher molecular weight cellulose aggregates, had a viscosity of 2.99±0.34 mPa·s. Despite being at a higher concentration, the medium composition containing cellulose oligomers had a viscosity equivalent to or lower than that of the medium composition containing cellulose nanofibers.

[0089] (4) Results of suspension culture

[0090] Figure 3 shows a microscopic image in the culture medium when cells were cultured for one day using a cellulose oligomer derived from Cellobios. Since the cells were observed in the culture medium without settling even after one day, it was found that by using a medium containing a cellulose oligomer, cells could be maintained and cultured in a three-dimensional manner in the culture medium.

[0091] In addition, as mentioned above, after culturing for one day, the medium was exchanged by centrifugation and redispersion. Microscopic images of the culture medium before and after the medium exchange are shown in Figure 4. As a result, it was found that the cells remained in the culture medium even after the medium exchange, and that the medium could be exchanged without adding new cellulose oligomers.

[0092] Figure 5 shows the microscopic image in the culture medium when cells were cultured for 5 days using glucose-derived and cellobiose-derived cellulose oligomers. In both cases where a dispersion of cellulose oligomers was used, it was observed that the cells proliferated and formed spheroids (aggregated clumps of cells), indicating that the cells were proliferating in a suspended state.

[0093] Figure 6 shows microscopic images of cells cultured for 2 days using a cellulose oligomer derived from Cellobios, followed by a culture without changing the medium for 2 days after the medium change, observed by fixing the observation site before and after culture. Since aggregated clumps of cells were observed at the same location before and after culture, it was found that the cells did not move during the culture process but remained in the same location and proliferated to form spheroids.

[0094] From the above results, it was clear that a dispersion of cellulose oligomers can be used for cell suspension culture.

[0095] (5) Results of recovering cultured cells

[0096] Figure 7 shows a microscopic image of the spheroids of cultured HeLa cells after recovery. Figure 7(A) shows the case using a cellulose oligomer derived from Cellobiose, and Figure 7(B) shows the case using a cellulose oligomer derived from glucose. After recovery, the microscopic image shows the spheroids settled on the bottom surface of the plate. The dispersion after culture was diluted and dispersed by pipetting, allowing for natural filtration. Therefore, by filtering using a mesh filter having a pore diameter (40 μm) that is smaller than the cultured spheroids and larger than the cellulose oligomer aggregate, only the spheroids could be separated and recovered on the filter.

[0097] The above describes several embodiments of the present invention. These embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments may be implemented in various other forms, and various omissions, substitutions, and modifications may be made within the scope of not departing from the essence of the invention. These embodiments, as well as the omissions, substitutions, and modifications thereof, are included within the scope equivalent to the invention described in the claims, just as they are included within the scope or essence of the invention.

Claims

Claim 1 A culture medium additive that is added to a culture medium to suspend cells and maintains the viscosity of the culture medium at 25°C at 0.50 mPa·s or more and 5.00 mPa·s or less, comprising a cellulose oligomer, wherein the cellulose oligomer is represented by the following general formula (1). In the formula, A represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms, and n represents an average degree of polymerization of 5 to 20. Claim 2 delete Claim 3 delete Claim 4 A culture medium composition capable of suspending and culturing cells, having a viscosity of 0.50 mPa·s or more and 5.00 mPa·s or less at 25°C, comprising a cellulose oligomer, wherein the cellulose oligomer is represented by the following general formula (1). In the formula, A represents a hydrogen atom or an alkyl group having 1 to 12 carbon atoms, and n represents an average degree of polymerization of 5 to 20. Claim 5 delete Claim 6 delete Claim 7 A method of culturing cells comprising culturing cells in a culture medium composition described in paragraph 4.

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