Porous cellulose particles and microcarriers for culture made from them

Abstract

Provided are: cellulose porous particles which have large surface areas on which cells can be attached, and on which cells can be adhered easily, and which rarely undergo the detachment of cells when the cells are cultured at a high density; and a microcarrier for culture use, which comprises the cellulose porous particles. The cellulose porous particles according to the present invention are composed of cellulose, in which the cellulose constituting the cellulose porous particles is modified by a cationic substituent and the cellulose constituting the cellulose porous particles has a polypeptide bound thereto.

Description

[Technical Field]

[0001] The present invention relates to porous cellulose particles used for cell culture, and to a culture microcarrier comprising the porous cellulose particles. [Background technology]

[0002] In recent years, there has been a demand for efficient mass cultivation of cells, tissues, microorganisms, etc. in the fields of vaccine and antibody drug production, regenerative medicine, etc. As a mass culture technique for the above cells, compared to the monolayer culture method using conventional flasks or roller bottles, which have a small culture surface area / volume ratio, the culture method using microcarriers can expand the culture surface area in a small volume while maintaining a high cell density per culture volume, and is therefore currently widely used in the culture of anchorage-dependent cells.

[0003] A widely used microcarrier is Cytodex1 (Cytiva), which has a charge capacity of approximately 1.5 mmol / g and is made of dextran particles modified with cationic diethylaminoethyl groups. However, because the particles lack a porous structure and cells are cultured only on the surface, proliferation capacity is limited. Furthermore, when cultured in a stirred reactor, shear stress is applied to the cells, causing them to fall off, significantly reducing culture efficiency.

[0004] To solve the above problems, porous microcarriers with large surface areas have been developed. Patent Document 1 below describes culturing using porous cellulose particles, which are characterized by interconnected pores with a pore size of approximately 2 μm or larger. When culturing using such porous particles, the large surface area allows efficient cell culture, and because the cells are positioned inside the particles, they are less susceptible to shear stress during agitation culture. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2-208331 [Patent Document 2] Japanese Patent Application Publication No. 5-252941 Summary of the Invention [Problem to be solved by the invention]

[0006] In the culture microcarrier using porous cellulose particles described in Patent Document 1, cells are rapidly adsorbed to the surface of the porous microcarrier by introducing a charge to the carrier surface in the early stages of culture when substrate adhesion between the microcarrier and cells is underdeveloped. However, some cell types have poor resistance to shear stress, which leads to cell detachment in the early stages of culture and reduced proliferation. When proliferation is low in the early stages of culture, extended culture is sometimes used to obtain a certain amount of cells or substances produced by the cells. However, with certain cells, long-term culture can result in a decrease in cell and protein activity. While there are methods for further increasing the surface charge to increase adhesion and prevent detachment, there is a problem that increasing the surface charge too much can inhibit proliferation in some cell types. Therefore, a method for preventing detachment while suppressing charge is needed.

[0007] To address these issues, the use of cell adhesion factors has been proposed as a means of promoting cell adhesion to particle surfaces. Cell adhesion factors are proteins that specifically bind to cell surface receptors thought to regulate cell adhesion. For example, Patent Document 2 presents a porous network structure coated with a peptide containing the amino acid sequence ArgGlyAsp (RGD) as a cell adhesion factor. However, the substrate described in Patent Document 2 is large and non-spherical, requiring a large stirring force to uniformly suspend and disperse the carrier in the medium during agitation culture. This results in high shear stress, which can lead to the detachment of cells placed on the surface before the adhesion effect that the adhesion factor should achieve is realized. Furthermore, the use of polyethyleneimine, which has a high charge density as a cationic substituent, makes it difficult to introduce an appropriate charge, resulting in growth inhibition in certain cells. In addition, polyethyleneimine is also used as a spacer for peptide conjugation, but because it is a branched polymer, it is prone to local imbalances in the charge and peptide modification levels, making it difficult to orient the peptide in the optimal direction, resulting in reduced cell adhesion. For these reasons, the method of Patent Document 2 is prone to cell dropout depending on the cell type and culture conditions, making it impossible to grow cells efficiently.

[0008] In view of the current state of the art described above, the problem to be solved by the present invention is to provide porous cellulose particles that have excellent cell adhesion, little cell dropout even when cultured at high density, and are capable of highly efficient cell proliferation, and a culture microcarrier made from such porous cellulose particles. [Means for solving the problem]

[0009] As a result of extensive research and experimentation to solve these problems, the inventors unexpectedly discovered that by introducing cell adhesive peptides into porous cellulose particles modified with cationic substituents, cells are less likely to fall off even when cultured at high density, and cell proliferation, particularly in the early stages of culture, is excellent. Based on this finding, the present invention was completed.

[0010] That is, the present invention is as follows. [1] A porous cellulose particle composed of cellulose, wherein the cellulose constituting the porous cellulose particle is modified with a cationic substituent, and the cellulose constituting the porous cellulose particle has a polypeptide bound to the cellulose. [2] The cellulose porous particles according to [1], wherein the porous particles have a charge capacity of 0.05 mmol / g or more and 5.0 mmol / g or less. [3] The cellulose porous particle according to [1] or [2], wherein at least a portion of the polypeptide is a cell adhesive peptide. [4] The porous cellulose particle according to any one of [1] to [3] above, wherein at least a part of the polypeptide is a peptide having the amino acid sequence of ArgGlyAsp(RGD). [5] The porous cellulose particles according to any one of [1] to [4], wherein the amount of the polypeptide carried is 10 nmol / g or more and 1 mmol / g or less per 1 g of the cell culture support (dry porous cellulose particles). [6] The porous cellulose particles according to any one of [1] to [5], wherein the amount of the polypeptide carried is 1.1 μmol / g or more and 1 mmol / g or less per 1 g of the cell culture support (dry porous cellulose particles). [7] The cellulose porous particle according to any one of [1] to [6] above, wherein the polypeptide has 5 to 30 amino acid residues. [8] The cellulose porous particles have a specific surface area of ​​0.3 m when dried. 2 / g or more 4.4m 2 The cellulose porous particles according to any one of the above [1] to [7], wherein the cellulose porous particles have a molecular weight of 1 / g or less. [9] The cellulose porous particles according to any one of [1] to [8], wherein the cellulose has a plurality of pores with an average pore size of 5 μm or more and 100 μm or less.

[10] The cellulose porous particle according to [9], wherein the pores form a continuous pore structure in which adjacent pores are interconnected by openings in the membrane that separate them.

[11] The cellulose porous particles according to any one of [1] to

[10] , which have an average particle size of 100 μm or more and 1000 μm or less.

[12] The cellulose porous particle according to any one of [1] to

[11] , wherein the cationic substituent is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

[13] The cellulose porous particle according to

[12] , wherein the cationic substituent is a tertiary amino group.

[14] The cellulose porous particle according to any one of [1] to

[13] above, wherein the polypeptide is bound to the cellulose via a spacer, and the chain length of the spacer is 5 atoms or more and 20 atoms or less.

[15] A microcarrier for culture, comprising the porous cellulose particles according to any one of [1] to

[14] above. [Effects of the Invention]

[0011] When the porous cellulose particles of the present invention are used as microcarriers, which are support bodies for cell culture, they can efficiently adsorb cells even in the early stages of culture when intercellular adhesion to the substrate is not yet fully developed. Furthermore, even when cultured at high density, cell detachment from the particles due to shear stress can be reduced, allowing cells to proliferate efficiently. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail. One embodiment of the present invention is a porous cellulose particle composed of cellulose, wherein the cellulose constituting the porous cellulose particle is modified with a cationic substituent and has a polypeptide bound to the cellulose.

[0013] The type of cellulose constituting the cellulose porous particles of this embodiment is not particularly limited, but known celluloses such as cuprammonium regenerated cellulose, viscose regenerated cellulose, cotton, pulp, and polynosic can be used, and cuprammonium regenerated cellulose and viscose regenerated cellulose are preferred, and cuprammonium regenerated cellulose is more preferred. The density of cellulose is 1.5 g / cm 3 This is the preferred density for materials used in microcarriers. Materials such as metals with a density of 2.0 or higher require a stronger stirring force to suspend the cells, which increases shear stress and damages the cells, making them unsuitable for agitated culture. Materials with a density of less than 1.0 float on the water surface, exposing the cells to air and making them unsuitable for agitated culture. Because cuprammonium regenerated cellulose has a low degree of crystallinity, it is highly reactive to various functional group modifications, making it possible to homogeneously modify the spacers and cationic functional groups necessary for the introduction of cell adhesion peptides, even in highly dense, highly porous particles. Furthermore, cuprammonium regenerated cellulose has a lower zeta potential than standard crystalline cellulose. This reduces interactions with cell adhesion peptides, such as electrostatic adsorption or repulsion, allowing for the homogeneous introduction of peptides into the porous interior. Furthermore, the peptides are arranged three-dimensionally on the surface of the microcarrier, enabling contact with cells in the appropriate orientation.

[0014] The shape of the porous cellulose particles of this embodiment is preferably spherical, more preferably having a sphericity of 0.70 to 1.00, and even more preferably 0.90 to 1.00. In this specification, the term "sphericity" refers to a value representing the shape of a particle. The porous particles are swollen with water, excess water is removed, and the projected area of ​​the particle as seen in an optical microscope image is measured. The value is defined as the ratio of the circumference of a circle having the same area to the actual circumference of the particle as seen in an electron microscope image. While this measurement method only views each particle in a plane, using the average value of at least 100 particles allows for consideration of variations in the observation direction, resulting in a three-dimensional representation of the particle's sphericity. The closer this value is to 1.00, the more spherical the particle's shape is; a perfect sphere is rated at 1.00. In spherical porous microcarriers, the internal liquid permeability is not anisotropic, so cell adhesion peptides can be uniformly modified even inside the particle, allowing cells to spread uniformly inside the particle. Furthermore, compared to other shapes such as cubes, buoyancy can be achieved with less stirring force, which reduces shear stress and prevents cells from falling out.

[0015] The lower limit of the average particle size of the porous cellulose particles of this embodiment is preferably 100 μm or more, more preferably 200 μm or more. An average particle size greater than 100 μm increases the number of cells retained per microcarrier, facilitating separation from the culture medium. The upper limit of the average particle size of the porous cellulose particles of this embodiment is preferably 1000 μm or less, more preferably 500 μm or less, and even more preferably 400 μm or less. An average particle size smaller than 1000 μm ensures sufficient supply of nutrients and oxygen to cells in the center of the microcarrier, resulting in a favorable culture environment. Furthermore, an average particle size smaller than 1000 μm reduces the stirring force required to suspend and disperse the microcarriers, thereby reducing shear stress. Therefore, cell detachment is less likely to occur, making the particles suitable for high-density culture. In this specification, the term "average particle size" refers to the average particle size measured by swelling porous particles with water, removing excess water, and measuring 20 or more particles using an optical microscope at an appropriate magnification.

[0016] The lower limit of the pore size of the porous cellulose particles of this embodiment is preferably an average pore size of 5 μm or more, more preferably 10 μm or more. By setting the average pore size to 5 μm or more, the degree of freedom of movement of cells and culture medium within the microcarrier is increased. The upper limit of the average pore size is preferably 100 μm or less, more preferably 80 μm or less, and even more preferably 50 μm or less. An average pore size smaller than 100 μm ensures a sufficient surface area for cell proliferation, resulting in efficient cell proliferation. In this specification, the term "average pore size" refers to the average pore size obtained by swelling porous particles with an ionic liquid, removing excess ionic liquid, and capturing a two-dimensional image at a resolution of 0.54 μm / pixel using a high-resolution 3D X-ray microscope, analyzing the image using image analysis software (Image J), ​​and measuring the intermembrane distance for three or more particles. The pore size is defined as the size of the largest circle that can fit inside the space of the porous particles in the two-dimensional image, as defined in the Thickness method.

[0017] The lower limit of the surface area (specific surface area) per 1 g of the cellulose porous particles of this embodiment when dried is 0.3 m 2 / g or more is preferable, and 0.6m 2 / g or more, more preferably 0.8m 2 The surface area required for cell culture is 0.3 m / g or more from the viewpoint of efficient cell proliferation. 2 The upper limit of the specific surface area is preferably 4.4 m / g or more. 2 / g or less, and 2 / g or less is more preferable, and 2.0m 2 / g or less is more preferable, and 1.7m 2 The specific surface area is particularly preferably 4.4 m / g or less, from the viewpoint of not reducing the charge per unit area, the amount of peptide, not reducing the cell adhesion rate, and further preventing cell detachment due to shear stress when cultured at high density. 2 / g or less. In this specification, the term "specific surface area" refers to the surface area per 1 g of dry weight measured by the BET method.

[0018] In this specification, the cellulose constituting the porous cellulose particles is modified with a cationic substituent. The cationic substituent is preferably at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group, more preferably a primary amino group or a tertiary amino group, and even more preferably a diethylaminoethyl group or a 3-amino-2-hydroxypropyl group. In this embodiment, when a tertiary amino group is used, it functions as a cationic functional group, while when a primary amino group is used, it functions as a condensation site with the spacer in addition to its function as a cationic functional group. Using a small molecule with little steric hindrance, such as a diethylaminoethyl group or a 3-amino-2-hydroxypropyl group, as the cationic substituent makes it possible to control the amount of cations within the optimal charge range and achieve uniform modification. On the other hand, when a polymer with a high charge density, such as polyethyleneimine, is used as the cationic substituent, it is difficult to control the amount of cations introduced, which may result in growth inhibition in certain cells.

[0019] Unlike particles such as polystyrene and gelatin, the charge capacity of the porous cellulose particles of this embodiment must take into account the influence of hydroxyl groups contained in the cellulose substrate. Furthermore, the intramolecular hydrogen bonding and crystallinity are significantly different from those of polysaccharide particles such as dextran particles, so this must also be taken into account. The lower limit of the charge capacity of the porous cellulose particles of this embodiment is preferably 0.01 mmol / g or more, more preferably 0.05 mmol / g or more, more preferably 0.5 mmol / g or more, more preferably 0.7 mmol / g or more, more preferably 0.9 mmol / g or more, more preferably 1 mmol / g or more, and particularly preferably 2 mmol / g or more. The upper limit of the charge capacity is preferably 5.0 mmol / g or less, more preferably 3.0 mmol / g or less. A charge capacity higher than 0.05 mmol / g allows for efficient cell adsorption in the early stages, while a charge capacity lower than 5.0 mmol / g is less likely to inhibit cell growth in the later stages of growth. In this specification, the term "charge capacity" refers to the average charge capacity measured for three or more samples obtained by adsorbing chlorine to the amine in the porous particles using hydrochloric acid, rinsing the adsorbed chlorine with a sodium sulfate solution, and then titrating the solution with silver nitrate using potassium chromate as an indicator.

[0020] A polypeptide is bound to the cellulose constituting the porous cellulose particles of this embodiment. The sequence of such a polypeptide preferably contains a peptide that is the active site of a cell adhesion factor. For example, there is the amino acid sequence represented by ArgGlyAsp (RGD) derived from fibronectin. The RGD peptide is a cell adhesion active sequence common to many cell adhesion proteins and effectively promotes the formation of adhesion between the microcarrier and the cell substrate. Other cell adhesive polypeptide sequences, represented by single-letter amino acid abbreviations, include, but are not limited to, amino acid sequences such as PHSRN and KNEED derived from fibronectin, YIGSR derived from laminin, and FHRRIKA derived from heparin. The molecular structure of the polypeptide chain of this embodiment is not particularly limited and may be linear, cyclic, or branched. Two or more peptides can be used in combination, but preferably, the polypeptide contains one or more peptides containing the amino acid sequence represented by ArgGlyAsp (RGD).

[0021] The number of amino acid residues in the polypeptide can be from 5 to 1000, but is preferably from 5 to 30. A peptide chain of fewer than 5 residues reduces the physiological activity of the peptide, while a peptide chain of 30 or more residues becomes intricately entangled, and the sequence acting as the adhesion factor becomes embedded inside the peptide molecule, making it difficult for cells to recognize it.

[0022] The lower limit of the content of the polypeptide is preferably 10 nmol / g or more, more preferably 100 nmol / g or more, more preferably 1 μmol / g or more, more preferably 1.5 μmol / g or more, even more preferably 3 μmol / g or more, and particularly preferably 10 μmol / g or more, per 1 g of the cell culture support (dry porous cellulose particles). The upper limit of the content of the polypeptide is preferably 1 mmol / g or less, more preferably 500 μmol / g or less, more preferably 200 μmol / g or less, and particularly preferably 100 μmol / g or less, per 1 g of the cell culture support (dry porous cellulose particles). The lower limit of the content per unit surface area of ​​the culture support is preferably 1 pmol / cm. 2More preferably, 10 pmol / cm 2 More preferably, 100 pmol / cm 2 More preferably, 150 pmol / cm 2 More preferably, 300 pmol / cm 2 More preferably, 1 nmol / cm 2 The upper limit of the content per unit surface area of ​​the cultivation carrier is 100 nmol / cm 2 Preferably less than 50 nmol / cm 2 or less, more preferably 20 nmol / cm 2 and particularly preferably 10 nmol / cm 2 The following results are obtained. If the polypeptide content is too low, adhesion and extensibility cannot be improved, while if the content is too high, steric hindrance makes it difficult to orient the polypeptide in the optimal direction, resulting in reduced cell extensibility. When culturing cells on microcarriers, stirring is required to ensure the microcarriers remain suspending, and cells are subjected to shear stress due to shear. Even under such conditions, good cell adhesion, proliferation, and viability are achieved when the polypeptide amount and charge capacity are within the above ranges. The polypeptide content in this embodiment was calculated by measuring the amount of fluorescent molecule (RB-PEG-SH, molecular weight 500, Biochempeg) introduced that can bind to the maleimide spacer group using a fluorometer. More specifically, the amount introduced was determined by calculating the unreacted fluorescent molecule remaining in the solution after the reaction from the fluorescence intensity of the supernatant of the reaction solution, and subtracting this value from the fluorescent molecule added to the reaction.

[0023] The polypeptide can be introduced by chemical bonding or physical adsorption, but a preferred method is to introduce it to cellulose via a spacer via chemical bonding. As used herein, the term "spacer" refers to a molecule that bridges the gap between the polypeptide having a cell adhesion peptide sequence and the porous cellulose particle. In this embodiment, the spacer is preferably a hydrophilic spacer containing an oxygen atom or a nitrogen atom, with 3-maleimidopropionic acid being particularly preferred. A simple alkyl linker increases the hydrophobicity of the surface, reducing the hydrophilicity of cellulose and decreasing its affinity with the cell culture medium. On the other hand, a highly hydrophilic linker such as PEG inhibits the adsorption of proteins necessary for cell adhesion, thereby reducing cell adhesion. The spacer preferably has a portion with rotational freedom and a portion with sterically controlled rigidity. A molecule having an alkyl group and a cyclic skeleton is more preferable, and 3-maleimidopropionic acid is particularly preferable. Because it has a cyclic skeleton with high steric hindrance and an alkyl group with high rotational freedom, the peptide chain can be positioned in a space with less steric hindrance, making it easier for cells to recognize the peptide chain.

[0024] The chain length of the spacer is not limited, but is preferably 5 to 20 atoms, more preferably 5 to 15 atoms. The chain length of the spacer refers to the number of atoms constituting the spacer when the shortest distance is taken between the bond between the spacer and the porous cellulose particle and the bond between the spacer and the polypeptide. For example, but not limited to, the constituent atoms may include carbon atoms, oxygen atoms, nitrogen atoms, sulfur atoms, silicon atoms, etc. If the spacer chain length is less than 5 atoms, the peptide cannot be maintained in the appropriate orientation, resulting in reduced recognition by cells and reduced cell adhesion and extensibility. On the other hand, if the spacer chain length exceeds 15 atoms, the chain length reduces the efficiency of introduction into the porous interior, and similarly reduces cell adhesion and extensibility.

[0025] The porous cellulose particles of this embodiment can be produced by a method in which a solution containing dissolved cellulose is formed into a desired shape while being cooled to a temperature below the solidification temperature of the solution to freeze it, and then the solvent is extracted and removed or the dissolving ability is lost. In general, the freezing temperature is preferably not set to a temperature more than 40°C lower than the freezing temperature of the solvent, and is usually selected in the range of 0 to 20°C lower than the freezing temperature.

[0026] The frozen cellulose or cellulose derivative solution is then subjected to extraction and removal of the solvent dissolving the cellulose or cellulose derivative, or to reducing its dissolving power (hereinafter, these processes are collectively referred to as "solvent removal, etc."), resulting in a solidified porous cellulose body. The conditions for solvent removal, etc., are not particularly limited. Typically, the frozen body can be quickly placed in a coagulation bath or regeneration bath, but it is preferable and recommended that the coagulation bath or regeneration bath be kept below the freezing point of the solution. However, in the case of cellulose derivatives, a cellulose regeneration process is required, and this regeneration is carried out simultaneously with or sequentially (i.e., after solvent removal, etc.). The regeneration itself can be carried out by conventional methods. Using the above-mentioned production method, there is no need to add foreign substances such as porosifying agents to the polymer solution during production, making it easy to produce minute, uniformly sized droplets, and particle size can be freely controlled. The size and shape of the pores are essentially determined by the size and shape of the solvent crystals formed when the solvent in the solution freezes and solidifies. Therefore, the shape and diameter of the pores can be adjusted by changing the type of polymer solution, the freezing and solidification conditions such as the temperature.

[0027] In this specification, the porous cellulose particles can be granulated cellulose particles as they are, but they are preferably crosslinked before use. While there are no limitations on the crosslinking method, any crosslinking agent with two or more functional groups capable of reacting with the hydroxyl groups of cellulose can be used to crosslink the molecules of the porous cellulose. Examples include bifunctional organic substances. Examples include XRZ-type compounds (where R represents an aliphatic residue containing a carbon atom, and X and Z are various halogens, epoxy groups, etc., bonded to the carbon atoms of the aliphatic residue) that react relatively easily in the presence of an alkaline reactant. Examples of bifunctional compounds suitable for the above reaction include epichlorohydrin, dichlorohydrin, 1,2- or 3,4-diepoxybutane, bisepoxypropyl ether, ethylene glycol-bisepoxypropyl ether, 1,4-butanediol-bisepoxypropyl ether, and closely related compounds, but are not limited to these. Crosslinking allows the crystalline structure of cellulose to be maintained even when modified with many cationic substituents, thereby keeping the particle structure stable.

[0028] In the production of the porous cellulose particles of this embodiment, the method for modifying the cellulose constituting the porous cellulose particles with a cationic substituent is not particularly limited, but multiple methods are possible, such as direct modification to hydroxyl groups or modification via a functional group having an epoxy group. For the introduction of tertiary amino groups, a method using 2-(diethylamino)ethyl chloride hydrochloride or the like to directly modify the hydroxyl groups of cellulose is preferred. Because cellulose contains many hydroxyl groups, direct modification of the hydroxyl groups makes it possible to modify the particles with a large number of cationic tertiary amino groups uniformly, even within the particles. For the introduction of primary amino groups, a method using ammonia via a functional group having an epoxy group is preferred. Because epoxy groups are electrically neutral molecules with little steric hindrance, it is possible to uniformly introduce a large number of primary amino groups uniformly within the particles, even after the introduction of cationic functional groups.

[0029] In the production of porous cellulose particles according to this embodiment, the method for introducing a polypeptide into the cellulose constituting the porous cellulose particles is not particularly limited. However, a preferred method involves introducing the polypeptide via chemical bonding, and a more preferred method involves reacting a peptide having a thiol group with a spacer having a maleimide group to bind the peptide to the cellulose. Specifically, the polypeptide can be introduced by adding a microcarrier having a maleimide group to an aqueous solution containing the polypeptide and stirring the mixture. When the peptide is introduced by physical adsorption, there is a risk of the polypeptide falling off during culture agitation. However, when the peptide is introduced via chemical bonding, the polypeptide can be retained on the surface even during agitation. Maleimide groups react specifically and irreversibly with peptides having a thiol group. The reaction proceeds under mild conditions in an aqueous solution of pH 6.5 to 7.5, allowing modification without affecting the molecular or particle structure of the microcarrier. Furthermore, the addition of a condensing agent or the like that would affect the culture is not necessary, and peptides can be introduced without causing cytotoxicity.

[0030] The porous cellulose particles of this embodiment can be used as microcarriers for culture. In this specification, "microcarriers for culture" refers to microcarriers used to attach and grow the culture target. During culture, the cells themselves may be the target of production, or a virus for vaccine production or the like may be the target cell growth product. In such cases, appropriate cells may be selected as the host. Depending on the purpose, it may be preferable to use genetically modified cells into which a production gene for the target cell growth product has been introduced. Regarding culture, for example, suspension culture and static culture may be used, but the use is not limited to these.

[0031] The cells used for culturing include, but are not limited to, fungi, yeast, plant cells, animal cells including fish and insects, and are preferably primate or human stem cells. Human stem cells include, but are not limited to, dental pulp stem cells, neural stem cells, skin stem cells, hematopoietic stem cells, mesenchymal stem cells, mammary stem cells, and endothelial stem cells. Primate or human stem cells are highly anchorage-dependent and interact strongly with cell adhesive peptides, making high-density culturing possible when using a culture microcarrier made of the porous cellulose particles of this embodiment. [Example]

[0032] EXAMPLES The present invention will be specifically explained below with reference to Examples and Comparative Examples, but the present invention is not limited to the Examples alone. The methods for measuring the physical properties of the structure will be described below. (1) Charge capacity Weigh out 5.0 g of the wet sample and, using a funnel and a suction filter, add 30 mL of 0.3 N hydrochloric acid solution twice, and then add 10 mL of -4 The solution was washed with 50 mL of 10% aqueous hydrochloric acid solution four times in sequence. Then, using a funnel and a suction filter, the solution was washed with 50 mL of 10% aqueous sodium sulfate solution four times, and the filtrate was collected. 2 mL of 0.1 N aqueous potassium chromate solution was added to the collected filtrate and stirred. Then, the solution was titrated with 1 N aqueous silver nitrate solution to determine the titer A (mL). Separately, the sample that had been washed with a 10% aqueous sodium sulfate solution in the above-mentioned process was washed four times with 50 mL of pure water, dried at 105°C for 15 hours or more, and weighed on a balance to determine the dry weight B (g). The charge capacity was calculated using the titer A (mL) measured above and the dry weight B (g) of the sample according to the following formula (1) (N=3). Charge capacity (mmol / g) = 1 (N) (mol / L) × A (mL) / B (g)...Equation (1)

[0033] (2) Polypeptide content Polypeptide content was assessed using a thiolated fluorescent molecule (RB-PEG-SH, molecular weight 500, Biochem PEG) with equivalent reactivity to the spacer. 1.0 g of sample was added to a reaction solution (100 g of purified water, 169.8 mg of WSCD·HCl, 282.4 mg of N-Hdroxysuccinimide (NHS), and 49.4 mg of 3-maleimidopropionic acid) adjusted to pH 6, incubated at 25°C for 24 hours, and then washed. 5 mL of 200 μM thiolated fluorescent molecule was added to 5 mg of sample and incubated for 24 hours. The incorporation rate was determined using a fluorometer as described below. The concentration (C) of the thiolated fluorescent molecule supernatant after 24 hours of incubation was determined by measuring the fluorescence intensity at 581 nm using an ARVO X3 fluorometer (PerkinElmer) with an excitation light wavelength of 554 nm. Here, the amount of RB-PEG-SH introduced into the sample was calculated by the following formula (2) using the concentration C (μmol / L) of the supernatant solution of the thiolated fluorescent molecule after the reaction (N=3). Amount of RB-PEG-SH introduced (μmol / g)=(200-C)(μmol / L)×5mL / 5mg…Equation (2)

[0034] [Examples 1 to 13, 16, and 17] A 500 mL solution of cellulose-copper-ammonium ammonium salts (2% cellulose, 1.2% copper, and 4% ammonia) with a viscosity of 2 poise at 5°C, prepared using purified linters, was sprayed with nitrogen gas using a two-fluid nozzle and stirred into 10 L of silicone oil (SH200, 1.5cs, Toray Industries, Inc.) cooled to -40°C. The silicone oil was heated to -20°C and stirred for 20 minutes, after which 10 L of 40% sulfuric acid cooled to -35°C was added. After adding the sulfuric acid, stirring was continued for 8 hours, and the mixture was heated to 20°C. The silicone oil was removed, and the remaining material was extracted and washed with water to obtain porous cellulose particles. Using a classification sieve, porous cellulose particles with an average particle size of 240 μm and an average pore size of 30 μm were obtained. Next, 20.0 g of particles were weighed out and added to a reaction solution (pure water: 217.30 g, anhydrous sodium sulfate: 88.83 g, sodium dodecyl sulfate: 0.016 g, 2-(diethylamino)ethyl chloride hydrochloride aqueous solution (50%): 11.08 g in Examples 1 to 6 and 12, and 13, 0.01 g in Example 7, 2.32 g in Examples 8 and 16, 22.2 g in Example 9, 44.4 g in Example 10, and 220 g in Examples 11 and 17) and stirred. Thereafter, a reaction initiation liquid (48% aqueous sodium hydroxide solution: 5.33 g in Examples 1 to 6, 12, and 13, 0.005 g in Example 7, 1.94 g in Examples 8 and 16, 10.6 g in Example 9, 21.3 g in Example 10, and 110 g in Examples 11 and 17) was added and gently stirred, followed by reaction with stirring for 1 hour in a thermostatic bath at 70°C. After 1 hour, 36% hydrochloric acid was added to neutralize the mixture until the pH reached 7, and the sample was washed three times with 8000 mL of pure water by suction filtration to obtain cationic porous cellulose particles.

[0035] Next, the sample was added to a reaction solution (259.74 g of purified water, 0.016 g of sodium dodecyl sulfate, 37.30 g of epichlorohydrin, and 12.65 g of 48% aqueous sodium hydroxide solution) and reacted for 1 hour with stirring in a constant temperature bath at 60°C. After 1 hour, the sample was washed three times with 8000 mL of purified water by suction filtration, and then the reaction solution (200 g of 23% aqueous ammonia solution) was added and reacted for 1 hour with stirring in a constant temperature bath at 40°C. After 1 hour, the sample was washed three times with 8000 mL of purified water by suction filtration, yielding cationic porous cellulose particles with primary and tertiary amino groups. Next, 1.0 g of the particles was added to a reaction solution (pure water: 100 g, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD·HCl): 169.8 mg, N-Hrdroxysuccinimide (NHS): 282.4 mg, 3-maleimidopropionic acid: 49.4 mg) adjusted to pH 6, and the mixture was reacted at 25°C for 24 hours. After washing, maleimide-group-introduced porous cellulose particles were obtained. Next, 78 mg of particles were added with peptide reaction solution (Example 1: 5 ml of aqueous solution with a CRGDS peptide concentration of 40 nM, Example 2: 5 ml of aqueous solution with a CRGDS peptide concentration of 200 nM, Example 3: 5 ml of aqueous solution with a CRGDS peptide concentration of 2 μM, Example 4: 5 ml of aqueous solution with a CRGDS peptide amount of 20 μM, Example 5: 5 ml of aqueous solution with a CRGDS peptide concentration of 200 μM, Example 6: 5 ml of aqueous solution with a CRGDS peptide concentration of 3 mM, Examples 7 to 11: 5 ml of aqueous solution with a CRGDS peptide concentration of 200 μM, Example 12: 5 ml of aqueous solution with a CGGEGYGEGYIGSR peptide concentration of 200 μM, Example 13: 5 ml of aqueous solution with a CSTATISGLKPGVDYTITVYAVTGRGD peptide concentration of 200 μM, Example 16: 5 ml of aqueous solution with a CRGDS peptide concentration of 40 nM, Example 17: 5 ml of aqueous solution with a CRGDS peptide concentration of 40 nM) and reacted at 25°C for 24 hours. After the reaction, the particles were washed with PBS(-) to obtain porous cellulose particles into which the polypeptide had been introduced. The particle charge capacity and amount of polypeptide are shown in Table 1 below.

[0036] [Example 14] Porous cellulose particles were obtained in the same manner as in Example 5, except that glycidyltrimethylammonium chloride was used instead of the 2-(diethylamino)ethyl chloride hydrochloride aqueous solution (50%).

[0037] [Example 15] A reaction solution (1.0 g of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate, 1.5 ml of triethylamine, 250 ml of acetonitrile, and 250 ml of tetrahydrofuran) was added to 6.66 g of cationic cellulose particles prepared using the same method as in Example 1, converting the primary amino groups to azide groups. Next, 5 ml of a reaction solution containing a peptide concentration of 200 μM was added to 1.0 g of particles, and the reaction was carried out at 25°C for 24 hours. RGD peptide was introduced via click reaction using a peptide with a G(Propargyl)-RGDS sequence containing an amino acid Gly(Propargyl) residue with an alkyne group in the side chain. After the reaction, the particles were washed with PBS(-) to obtain porous cellulose particles with the polypeptide introduced.

[0038] The resulting particles were used to culture hDPSC-dental pulp stem cells (PT-5025, Lonza) for 10 days. The particles were added to DMEM + 10% FBS medium to obtain a 10 mg / mL particle suspension (600 μL). A dental pulp stem cell suspension (2.4 mL, 2.25 × 10 4 The cells were incubated at 37°C with 100 μM calcein-AM (Live / Dead Cell Staining Kit II, Protocell) at 37°C. After the specified number of days, the particles were sampled and washed three times with PBS(-). 3 mL of 1 μM Calcein-AM (Live / Dead Cell Staining Kit II, Protocell) was added and the cells were incubated for 15 minutes in the dark to stain the cells. After washing three times with PBS(-), the cells were irradiated with 488 nm excitation light using a confocal laser microscope (FV-3000, OLYMPUS). Fluorescence at wavelengths of 500-540 nm was observed and photographed using a 10X magnification lens in the Z-axis direction to a depth of 260 μm. The ratio of the fluorescence area to the particle area was calculated using analysis software (Image J) to evaluate the amount of cell attachment. The results are shown in Table 1 below.

[0039] Table 1 below shows the evaluation of adhesion at the initial stage of culture (amount of viable cells attached on the first day of culture), proliferation at the initial stage of culture (amount of proliferation on the fourth day relative to the amount of viable cells attached on the first day of culture), proliferation during the middle stage of culture (amount of viable cells attached on the seventh day of culture), and long-term viability (amount of viable cells attached on the tenth day of culture). Adhesion at the early stage of culture (first day of culture) was evaluated using the fluorescence area ratio of Comparative Example 1 as the standard: an area ratio of 150% or more was evaluated as ◎, an area ratio of 130% or more was evaluated as 〇, an area ratio of 100% or more was evaluated as △, and an area ratio of less than 100% was evaluated as ×. The proliferation rate at the initial stage of culture was evaluated by measuring the fluorescence area ratio on the fourth day of culture relative to the fluorescence area ratio on the first day of culture. An area increase ratio of 400% or more was evaluated as ◎, an area increase ratio of 300% or more was evaluated as 〇, an area increase ratio of 200% or more was evaluated as △, and an area increase ratio of less than 200% was evaluated as ×. The proliferation during the mid-culture period was evaluated by measuring the fluorescence area ratio on the 7th day of culture relative to the fluorescence area ratio on the 1st day of culture, with an area increase ratio of 800% or more marked as ◎, an area increase ratio of 600% or more marked as 〇, an area increase ratio of 400% or more marked as △, and an area increase ratio of less than 400% marked as ×. Long-term viability was evaluated based on the fluorescence area ratio on the 10th day of culture, using the fluorescence area ratio on the 10th day of culture in Comparative Example 1 as the standard. An area ratio of 400% or more was evaluated as ◎, an area ratio of 200% or more was evaluated as ○, an area ratio of 150% or more was evaluated as △, and an area ratio of less than 150% was evaluated as ×.

[0040] [Comparative Example 1] Cationic porous cellulose particles were prepared in the same manner as in Example 1, but the particles were obtained without introducing maleimide groups or polypeptides. The charge capacity and polypeptide amount of the particles are shown in Table 1 below. The cell proliferation rate was also evaluated in the same manner as in Example 1.

[0041] Comparative Example 2 Cellulose porous particles were prepared using the method described in Example 1, with only primary amino groups introduced, but no tertiary amino groups, and then maleimide groups and polypeptides were introduced to obtain particles. The particle charge capacity and polypeptide amount are shown in Table 1 below. Cell proliferation was also evaluated using the same method as in Example 1.

[0042] [Table 1] [Industrial Applicability]

[0043] When the porous cellulose particles of the present invention are used as a culture support (culture microcarrier), they can efficiently adsorb and spread cells even in the early stages of culture when intercellular substrate adhesion is underdeveloped. Furthermore, even when cultured at high density, cell detachment from the particles due to shear stress can be reduced, allowing cells to proliferate efficiently.

Claims

1. A porous cellulose particle composed of cellulose, wherein the cellulose constituting the porous cellulose particle has a polypeptide bound to the cellulose, and the cellulose constituting the porous cellulose particle is modified with a cationic substituent that does not function as a condensation site between the cellulose and a spacer.

2. 2. The cellulose porous particles according to claim 1, wherein the charge capacity of the cellulose porous particles is 0.05 mmol / g or more and 5.0 mmol / g or less.

3. The cellulose porous particle according to claim 1 or 2, wherein at least a portion of the polypeptide is a cell adhesive peptide.

4. The cellulose porous particle according to claim 1 or 2, wherein at least a portion of the polypeptide is a peptide having the amino acid sequence ArgGlyAsp (RGD).

5. The cellulose porous particles according to claim 1 or 2, wherein the amount of the polypeptide carried per gram of the cell culture support (dry porous cellulose particles) is 10 nmol / g or more and 1 mmol / g or less.

6. 3. The cellulose porous particle according to claim 1, wherein the amount of the polypeptide carried per gram of the cell culture carrier (dry porous cellulose particle) is 1.1 μmol / g or more and 1 mmol / g or less.

7. 3. The cellulose porous particle according to claim 1, wherein the polypeptide has 5 to 30 amino acid residues.

8. The cellulose porous particles have a specific surface area of ​​0.3 m when dried. 2 / g or more 4.4m 2 The cellulose porous particles according to claim 1 or 2, wherein the pore size is 1 / g or less.

9. The cellulose porous particle according to claim 1 or 2, wherein the cellulose has a plurality of pores with an average pore size of 5 μm or more and 100 μm or less.

10. 10. The cellulose porous particle according to claim 9, wherein the pores form a continuous pore structure in which the pores communicate with each other via openings in the membrane that separate adjacent pores.

11. The cellulose porous particles according to claim 1 or 2, having an average particle size of 100 μm or more and 1000 μm or less.

12. 3. The cellulose porous particle according to claim 1, wherein the cationic substituent is at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group.

13. The cellulose porous particle according to claim 12, wherein the cationic substituent is a tertiary amino group.

14. 3. The cellulose porous particle according to claim 1, wherein the polypeptide is bound to the cellulose via a spacer, and the chain length of the spacer is 5 atoms or more and 20 atoms or less.

15. A microcarrier for culture, comprising the porous cellulose particles according to claim 1 or 2.

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