Microcarriers for cell culture and methods for culturing cells

Abstract

This invention provides a microcarrier for cell culture that can suppress damage to microcarriers during the cell culture process and improve the efficiency of cell culture. [Solution] The microcarrier for cell culture according to the present invention comprises a base particle and a coating layer that covers the outer surface of the base particle, and has a breaking strength of 1000 mN or more.

Description

[Technical Field]

[0001] This invention relates to a microcarrier for cell culture. Furthermore, this invention relates to a method for culturing cells using the above-mentioned microcarrier for cell culture. [Background technology]

[0002] In research and development in academic fields, drug discovery, and regenerative medicine, animal cells from humans, mice, rats, pigs, cattle, and monkeys are used. Microcarrier-based cell culture methods are known.

[0003] Conventionally, microcarriers in which substrate particles are coated with an extracellular matrix (ECM) have been widely used as the above-mentioned microcarriers. For example, Patent Document 1 below describes a microcarrier having polystyrene particles and vitronectin arranged on the outer surface of the polystyrene particles.

[0004] Furthermore, microcarriers in which base particles are coated with synthetic resin are also known. For example, Patent Document 2 below describes a microcarrier comprising polystyrene particles and a synthetic resin layer disposed on the outer surface of the polystyrene particles. The synthetic resin layer contains a synthetic resin to which peptides are bound. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2011 / 017167A1 [Patent Document 2] WO2011 / 017050A1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Conventional microcarriers, such as those described in Patent Documents 1 and 2, use resin particles with a low degree of crosslinking, such as polystyrene particles, as base particles. As a result, conventional microcarriers may be damaged during the cell culture process. For example, in conventional microcarriers, collisions between microcarriers during cell culture or impacts during culture medium exchange can cause chipping or cracking. Such microcarrier damage is particularly likely to occur when culturing cells on a scale of several hundred liters or more using large-scale culture equipment.

[0007] If a microcarrier is damaged, it is difficult to separate the microcarrier fragments from the cells, and therefore, for example, such fragments may be present in cell preparations.

[0008] Furthermore, high cell culture efficiency is preferable.

[0009] The object of the present invention is to provide a microcarrier for cell culture that can suppress damage to microcarriers during the cell culture process and improve the efficiency of cell culture. The object of the present invention is also to provide a method for culturing cells using the above-mentioned microcarrier for cell culture. [Means for solving the problem]

[0010] In a broader sense, the present invention provides a microcarrier for cell culture (hereinafter sometimes abbreviated as "microcarrier") comprising a base particle and a coating layer covering the outer surface of the base particle, wherein the breaking strength is 1000 mN or more.

[0011] In a specific aspect of the microcarrier according to the present invention, the compression displacement curve obtained when a compression test is performed does not have an inflection point when the load is 700 mN or less.

[0012] In certain aspects of the microcarrier according to the present invention, the water absorption rate is 10% by weight or less.

[0013] In a specific aspect of the microcarrier according to the present invention, the coating layer contains a synthetic resin.

[0014] In a specific aspect of the microcarrier according to the present invention, the synthetic resin has a polyvinyl alcohol derivative skeleton or a poly(meth)acrylate skeleton.

[0015] In a specific aspect of the microcarrier according to the present invention, the coating layer contains a peptide moiety.

[0016] In a specific aspect of the microcarrier according to the present invention, the base material particles are resin particles.

[0017] In a specific aspect of the microcarrier according to the present invention, the base material particles contain a polymer of a monomer having an ethylenically unsaturated group.

[0018] In a specific aspect of the microcarrier according to the present invention, the polymer of the monomer having an ethylenically unsaturated group is an acrylic resin, a divinylbenzene polymer, or a divinylbenzene copolymer.

[0019] In a specific aspect of the microcarrier according to the present invention, the specific gravity is 1.0 g / cm 3 or more and 2.0 g / cm 3 or less.

[0020] In a specific aspect of the microcarrier according to the present invention, the average particle diameter is 100 μm or more and 1500 μm or less.

[0021] In a specific aspect of the microcarrier according to the present invention, the CV value of the particle diameter is 10% or less.

[0022] According to a broad aspect of the present invention, there is provided a method for culturing cells, which comprises a step of adhering cells to the above-described microcarrier for cell culture.

Effects of the Invention

[0023] The microcarrier for cell culture according to the present invention comprises a base particle and a coating layer covering the outer surface of the base particle, and has a breaking strength of 1000 mN or more. Because the microcarrier for cell culture according to the present invention has the above configuration, damage to the microcarrier during the cell culture process can be suppressed, and the efficiency of cell culture can be increased. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 is a schematic cross-sectional view showing a microcarrier for cell culture according to one embodiment of the present invention. [Figure 2] Figure 2 is a photograph of a damaged microcarrier observed in Comparative Example 1. [Modes for carrying out the invention]

[0025] The details of the present invention will be described below.

[0026] (Microcarriers for cell culture) The microcarrier for cell culture according to the present invention (hereinafter sometimes abbreviated as "microcarrier") comprises a base particle and a coating layer covering the outer surface of the base particle, and has a breaking strength of 1000 mN or more.

[0027] Since the microcarrier according to the present invention is equipped with the above configuration, damage to the microcarrier during the cell culture process can be suppressed, and the efficiency of cell culture can be increased.

[0028] Conventional microcarriers have relatively low strength. Therefore, with conventional microcarriers, collisions between microcarriers during cell culture or impacts during culture medium changes can cause chipping or cracking. Such microcarrier damage is particularly likely to occur when culturing cells using large-scale culture equipment, making it difficult to use conventional microcarriers in cell cultures on a scale of several hundred liters.

[0029] In contrast, the microcarriers according to the present invention have relatively high strength. Therefore, even if the microcarriers collide with each other during cell culture or are subjected to impact during culture medium exchange, the microcarriers are less likely to chip or break. Consequently, the microcarriers according to the present invention can reduce the risk of contamination by microcarrier fragments in cell preparations, for example.

[0030] Furthermore, the microcarrier according to the present invention can improve the efficiency of cell culture.

[0031] The microcarrier according to the present invention can be suitably used in cell cultures ranging from tens of milliliters to hundreds of liters or more.

[0032] Furthermore, since the microcarriers according to the present invention do not require the use of natural polymer materials such as extracellular matrix (ECM) as materials, they are inexpensive, have low lot-to-lot variability, and offer excellent safety.

[0033] From the perspective of suppressing microcarrier damage during the cell culture process, the breaking point strength of the above microcarriers is 1000 mN or higher. In other words, the above microcarriers do not have a breaking point strength of less than 1000 mN.

[0034] The breaking strength of the above microcarrier is preferably 1100 mN or more, more preferably 1200 mN or more, and even more preferably 1500 mN or more. If the breaking strength is above the lower limit, damage to the microcarrier during the cell culture process can be suppressed more effectively. There is no particular upper limit to the breaking strength of the above microcarrier. The breaking strength of the above microcarrier may be 10000 mN or less.

[0035] The fracture strength of the above microcarrier is the fracture strength when the microcarrier is compressed. The fracture strength of the above microcarrier is the fracture strength when the following compression test is performed. The fracture strength of the above microcarrier can be measured as follows.

[0036] A microcarrier compression test is performed using a microstrength evaluation test apparatus, applying a maximum test load of 2000 mN at 0.3 N / second to the smooth end face of a cylindrical diamond indenter (500 μm in diameter) at 25°C. The load at which the microcarrier breaks is defined as the breaking point strength of the microcarrier. As the microstrength evaluation test apparatus, for example, the "Microautograph MST-I" manufactured by Shimadzu Corporation is used.

[0037] The fracture strength of the above-mentioned microcarriers can be increased, for example, by using a resin with a high degree of crosslinking as the base particle material, using base particles containing fillers, or using a resin with a flexible molecular structure as the base particle material.

[0038] In the above-mentioned microcarriers, it is preferable that the compression displacement curve obtained during a compression test does not have an inflection point when the load is 700 mN or less. In this case, damage to the microcarriers during the cell culture process can be suppressed even more effectively.

[0039] The conditions for the compression test used to obtain the above-mentioned compression displacement curve are the same as those for the compression test used to determine the fracture strength. The above-mentioned compression displacement curve can be obtained as follows.

[0040] In the compression test of the microcarrier described above, the load value (mN) and compressive displacement (μm) are measured, and a compressive displacement curve showing the relationship between compressive displacement (x axis) and load value (y axis) is created. The point where the slope of the tangent line of the obtained compressive displacement curve changes from increasing to decreasing is defined as the inflection point. It is then confirmed whether the obtained compressive displacement curve has an inflection point when the load is 700 mN or less.

[0041] Furthermore, when a microcarrier is compressed, the slope of the tangent to the compression displacement curve tends to increase, but when a crack occurs in the microcarrier, the slope of the tangent to the compression displacement curve tends to decrease. Therefore, if the obtained compression displacement curve does not have an inflection point at loads of 700 mN or less, the inclusion of fragments generated from the microcarrier can be suppressed even more effectively.

[0042] The water absorption rate of the above microcarrier is preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 1% by weight or less. When the water absorption rate is below the above upper limit, the surface state of the microcarrier is less likely to change during cell adhesion, thus reducing the variability in the initial colonization rate after cell seeding. Furthermore, when the water absorption rate is below the above upper limit, cells are less likely to detach from the microcarrier in the culture medium. The lower limit of the water absorption rate of the above microcarrier is not particularly limited. The water absorption rate of the above microcarrier may be 0% by weight or more, or 0.001% by weight or more.

[0043] The water absorption rate of the above microcarriers can be measured as follows.

[0044] Prepare microcarriers by drying them in a 100°C oven for 8 hours. Leave 100.0 mg of these microcarriers for 24 hours in an environment with a temperature of 37°C and a relative humidity of 95% RH. Measure the weight of the microcarriers after this period. Calculate the water absorption rate of the microcarriers using the following formula.

[0045] Water absorption rate (weight %)=(W2-W1) / W1×100 W1: Weight of microcarriers before standing (mg) W2: Weight of microcarriers after standing (mg)

[0046] One method for reducing the water absorption rate of the above-mentioned microcarriers is to create a coating layer using a highly hydrophobic material.

[0047] The specific gravity of the above microcarriers is preferably 1.0 g / cm³. 3 More preferably 1.05 g / cm³ 3 More preferably 1.1 g / cm³ 3 Preferably 2.0 g / cm³ 3 More preferably, 1.5 g / cm³ 3 More preferably, 1.3 g / cm³ 3 The following applies: If the specific gravity is above the lower limit, the microcarriers will settle appropriately, improving the recovery efficiency. If the specific gravity is below the upper limit, the rotational performance of the agitator blades can be improved.

[0048] The specific gravity of the above microcarriers is measured using a hydrometer.

[0049] The average particle size of the above microcarriers is preferably 100 μm or more, more preferably 150 μm or more, even more preferably 200 μm or more, still more preferably 250 μm or more, particularly preferably 300 μm or more, preferably 1500 μm or less, more preferably 1000 μm or less, even more preferably 800 μm or less, still more preferably 700 μm or less, and particularly preferably 500 μm or less. The average particle size of the above microcarriers is preferably 100 μm or more and 1500 μm or less, more preferably 150 μm or more and 1000 μm or less, even more preferably 200 μm or more and 800 μm or less, still more preferably 250 μm or more and 700 μm or less, and particularly preferably 300 μm or more and 500 μm or less. If the above average particle size is above the lower limit, the cell culture efficiency can be further increased. If the above average particle size is below the upper limit, cell aggregates can be formed with a more uniform thickness on the surface of each microcarrier. Furthermore, if the average particle size is below the upper limit, the area on which cells can adhere can be further increased. With conventional microcarriers, the larger the average particle size, the more likely the microcarriers are to be damaged during the cell culture process. However, with the microcarriers according to the present invention, damage to the microcarriers during the cell culture process can be suppressed even if the average particle size is relatively large.

[0050] The particle size of the above-mentioned microcarrier refers to the diameter if the microcarrier is spherical, and if the microcarrier has a shape other than a perfect sphere, it refers to the diameter assuming it is a perfect sphere of a volume equivalent to that shape.

[0051] The average particle diameter of the above microcarriers is preferably the number-average particle diameter. The average particle diameter of the above microcarriers can be determined by observing 50 arbitrary microcarriers with an electron microscope or optical microscope and calculating the average value of the particle diameter of each microcarrier, or by using a particle size distribution analyzer. In observation with an electron microscope or optical microscope, the particle diameter of a single microcarrier is determined as the particle diameter at the equivalent diameter of a circle. In observation with an electron microscope or optical microscope, the average particle diameter at the equivalent diameter of a circle of any 50 microcarriers is approximately equal to the average particle diameter at the equivalent diameter of a sphere. In a particle size distribution analyzer, the particle diameter of a single microcarrier is determined as the particle diameter at the equivalent diameter of a sphere. It is preferable to calculate the average particle diameter of the above microcarriers using a particle size distribution analyzer.

[0052] The coefficient of variation (CV value) of the particle size of the above microcarriers is preferably 10% or less, more preferably 8% or less, even more preferably 5% or less, and particularly preferably 3% or less. When the coefficient of variation (CV value) is below the above upper limit, the uniformity of the sedimentation velocity can be increased, and the efficiency of cell culture can be further increased. The coefficient of variation (CV value) of the particle size of the above microcarriers may be 0% or more, 0.1% or more, 0.5% or more, or 1% or more. The coefficient of variation (CV value) of the particle size of the above microcarriers may be 0% or more and 10% or less, 0.1% or more and 8% or less, 0.1% or more and 5% or less, or 1% or more and 3% or less.

[0053] The coefficient of variation (CV value) of the particle size of the above microcarriers is calculated as follows.

[0054] CV value (%) = (ρ / Dn) × 100 ρ: Standard deviation of the particle size of the microcarrier Dn: Average particle size of microcarriers

[0055] Methods for reducing the coefficient of variation (CV value) of the particle size of the above-mentioned microcarriers include dry classification and wet classification.

[0056] The shape of the microcarrier described above is not particularly limited. The shape of the microcarrier may be spherical, or it may be a shape other than spherical, such as a flattened shape. Note that the spherical shape is not limited to a perfect sphere, but also includes a nearly spherical shape, for example, a shape with an aspect ratio (major axis / minor axis) of 1.5 or less.

[0057] The present invention will be described in detail below with reference to the drawings.

[0058] Figure 1 is a schematic cross-sectional view showing a microcarrier for cell culture according to one embodiment of the present invention.

[0059] The cell culture microcarrier 1 shown in Figure 1 comprises a base particle 2 and a coating layer 3 that covers the outer surface of the base particle 2. The coating layer 3 is positioned on the surface of the base particle 2 and is in contact with the surface of the base particle 2. The coating layer 3 covers the entire outer surface of the base particle 2. The breaking strength of the microcarrier 1 is 1000 mN or more.

[0060] Further details about microcarriers are described below.

[0061] In this specification, "(meth)acrylate" means either or both "acrylate" and "methacrylate," and "(meth)acrylic" means either or both "acrylic" and "methacrylic."

[0062] (base material particles) The material of the above-mentioned base particles is not particularly limited, as long as the breaking strength of the microcarriers is 1000 mN or more. The material of the above-mentioned base particles may be an organic material, an inorganic material, or both an organic and an inorganic material. The above-mentioned base particles may contain a resin, an inorganic filler, a resin and an inorganic filler, or may not contain a resin. The above-mentioned base particles may be resin particles or inorganic particles. It is preferable that the above-mentioned base particles contain a resin. From the viewpoint of further suppressing damage to microcarriers during the cell culture process, it is preferable that the above-mentioned base particles are resin particles. The material of the above-mentioned base particles may be used by one type only, or two or more types may be used in combination. The above-mentioned resin may be used by one type only, or two or more types may be used in combination.

[0063] Examples of the above-mentioned resins include polyolefin resins, acrylic resins, polycarbonates, polyamides, phenol formaldehyde resins, melamine formaldehyde resins, benzoguanamine formaldehyde resins, urea formaldehyde resins, phenolic resins, melamine resins, benzoguanamine resins, urea resins, epoxy resins, unsaturated polyester resins, saturated polyester resins, polyethylene terephthalate, polysulfones, polyphenylene oxides, polyacetals, polyimides, polyamide-imides, polyetheretherketones, polyethersulfones, divinylbenzene polymers, and divinylbenzene copolymers.

[0064] When the above-mentioned base material particles are resin particles, among the various resins available, a resin that can control the breaking point strength of the microcarriers to 1000 mN or more is preferably used.

[0065] The above resin is preferably a polymer of monomers having ethylenically unsaturated groups. The above base particles preferably contain a polymer of monomers having ethylenically unsaturated groups. In this case, the specific gravity and strength of the base particles can be well adjusted, and as a result, the specific gravity of the microcarriers can be adjusted to a suitable range, and the breaking strength of the microcarriers can be increased.

[0066] The monomer having the ethylenically unsaturated group described above preferably has two or more such groups. The base particles preferably contain a polymer of monomers having two or more ethylenically unsaturated groups. In this case, the specific gravity and strength of the base particles can be adjusted more effectively, and as a result, the specific gravity of the microcarriers can be adjusted to a suitable range, and the breaking strength of the microcarriers can be increased.

[0067] Examples of polymers of monomers having the above-mentioned ethylenically unsaturated group include acrylic resins, divinylbenzene polymers, and divinylbenzene copolymers. The above-mentioned monomers having the ethylenically unsaturated group may be used individually or in combination of two or more.

[0068] The polymer of the monomer having the ethylenically unsaturated group described above is preferably an acrylic resin, a divinylbenzene polymer, or a divinylbenzene copolymer. In this case, the specific gravity and strength of the base particles can be well adjusted, and as a result, the specific gravity of the microcarriers can be adjusted to a suitable range, and the breaking point strength of the microcarriers can be increased.

[0069] When the above-mentioned base particles contain a polymer of a monomer having an ethylenically unsaturated group, it is preferable that the polymer of the monomer having an ethylenically unsaturated group has a crosslinked structure. In this case, the specific gravity and strength of the base particles can be well adjusted, and as a result, the specific gravity of the microcarriers can be adjusted to a suitable range, and the breaking strength of the microcarriers can be increased.

[0070] Examples of methods for forming the above-mentioned crosslinked structure include: (1) a method of polymerizing a polymerizable component containing a monomer having two or more ethylenically unsaturated groups; and (2) a method of forming a crosslinked structure by reacting a polymer of monomers having ethylenically unsaturated groups with a crosslinking agent.

[0071] In the method described in (1) above, examples of monomers having two or more ethylenically unsaturated groups include divinylbenzene, polyfunctional (meth)acrylate, triallyl(iso)cyanurate, triallyl trimellitate, diallyl phthalate, and diallyl acrylamide. Only one of the monomers having two or more ethylenically unsaturated groups may be used, or two or more may be used in combination.

[0072] Furthermore, in the method of (1) described above, the polymerizable component may also include other monomers having an ethylenically unsaturated group. Examples of other monomers having an ethylenically unsaturated group include styrene, monofunctional (meth)acrylate, (meth)acrylic acid, acrylonitrile, vinyl chloride, and the like. Only one of the other monomers having an ethylenically unsaturated group may be used, or two or more may be used in combination.

[0073] Examples of polymers obtained by the method described in (1) above include copolymers of divinylbenzene and styrene, and copolymers of polyfunctional (meth)acrylate and monofunctional (meth)acrylate.

[0074] One example of the method described in (2) above is a polymerizable component containing a monomer having an ethylenically unsaturated group and a functional group containing active hydrogen in its molecule, which is polymerized to obtain a polymer, and then a crosslinking agent is used to crosslink the polymers.

[0075] Examples of functional groups containing the active hydrogen mentioned above include hydroxyl groups, carboxyl groups, amino groups, and phenol groups. Examples of monomers having an ethylenically unsaturated group and a functional group containing active hydrogen in their molecule include hydroxyl group-containing (meth)acrylate, (meth)acrylic acid, and amino group-containing (meth)acrylate. Only one of the above monomers having an ethylenically unsaturated group and a functional group containing active hydrogen in their molecule may be used, or two or more may be used in combination.

[0076] The above crosslinking agent is not particularly limited as long as it can react with the functional group containing the active hydrogen, and examples include polyfunctional isocyanate compounds and polyfunctional epoxy compounds. The above crosslinking agent may be used alone or in combination of two or more.

[0077] Furthermore, as long as the fracture strength of the resulting microcarriers is 1000 mN or more, the method for forming the cross-linked structure is not limited to these methods.

[0078] The above-mentioned substrate particles can be obtained, for example, by polymerizing the monomer having the above-mentioned ethylenically unsaturated group. The polymerization method is not particularly limited and includes known methods such as radical polymerization, ionic polymerization, polycondensation (condensation polymerization, condensation polymerization), addition condensation, living polymerization, and living radical polymerization. Another polymerization method is suspension polymerization in the presence of a radical polymerization initiator.

[0079] The above-mentioned base material particles may contain inorganic fillers. For example, by using inorganic fillers in combination with a resin with a low specific gravity, the specific gravity of the base material particles and microcarriers can be suitably increased.

[0080] Examples of the inorganic fillers mentioned above include carbon black, glass fillers, and metal fillers. Only one type of inorganic filler may be used, or two or more types may be used in combination.

[0081] In 100% by weight of the above base material particles, the content of the above resin is preferably 80% by weight or more, more preferably 90% by weight or more, still more preferably 95% by weight or more, further preferably 97% by weight or more, still further preferably 99% by weight or more, and most preferably 100% by weight (total amount). Note that, in 100% by weight of the above base material particles, the content of the above resin may be 100% by weight or less, or may be less than 100% by weight.

[0082] In 100% by weight of the above base material particles, the content of the polymer of the monomer having an ethylenically unsaturated group is preferably 80% by weight or more, more preferably 90% by weight or more, still more preferably 95% by weight or more, further preferably 97% by weight or more, still further preferably 99% by weight or more, and most preferably 100% by weight (total amount). Note that, in 100% by weight of the above base material particles, the content of the polymer of the monomer having an ethylenically unsaturated group may be 100% by weight or less, or may be less than 100% by weight.

[0083] When the above base material particles are the above inorganic particles, examples of the inorganic particles include graphite particles, glass particles, metal particles, and the like.

[0084] The specific gravity of the above base material particles is preferably 1.0 g / cm 3 or more, more preferably 1.05 g / cm 3 or more, still more preferably 1.1 g / cm 3 or more, preferably 2.0 g / cm 3 or less, more preferably 1.5 g / cm 3 or less, still more preferably 1.3 g / cm 3 or less, further preferably 1.2 g / cm 3 or less, particularly preferably 1.15 g / cm 3 or less. When the specific gravity is above the above lower limit and below the above upper limit, the specific gravity of the microcarrier can be adjusted to a suitable range.

[0085] The specific gravity of the above base material particles is measured using a true specific gravity meter.

[0086] The average particle size of the above substrate particles is preferably 100 μm or more, more preferably 150 μm or more, even more preferably 200 μm or more, still more preferably 250 μm or more, particularly preferably 300 μm or more, preferably 1500 μm or less, more preferably 1000 μm or less, even more preferably 800 μm or less, still more preferably 700 μm or less, and particularly preferably 500 μm or less. The average particle size of the above substrate particles is preferably 100 μm or more and 1500 μm or less, more preferably 150 μm or more and 1000 μm or less, even more preferably 200 μm or more and 800 μm or less, still more preferably 250 μm or more and 700 μm or less, and particularly preferably 300 μm or more and 500 μm or less. If the above average particle size is above the lower limit, the cell culture efficiency can be further increased. If the above average particle size is below the upper limit, cell aggregates can be formed with an even more uniform thickness on the surface of each microcarrier. Furthermore, if the average particle size is below the upper limit mentioned above, the surface area on which cells can adhere can be further increased.

[0087] The particle diameter of the above-mentioned base material particles refers to the diameter if the base material particles are spherical, and if the base material particles have a shape other than a perfect sphere, it refers to the diameter assuming they are spherical to the extent of their volume.

[0088] The average particle diameter of the above-mentioned substrate particles is preferably the number-average particle diameter. The average particle diameter of the above-mentioned substrate particles can be determined by observing 50 arbitrary substrate particles with an electron microscope or optical microscope and calculating the average value of the particle diameter of each substrate particle, or by using a particle size distribution analyzer. In observation with an electron microscope or optical microscope, the particle diameter of a single substrate particle is determined as the particle diameter at the equivalent diameter of a circle. In observation with an electron microscope or optical microscope, the average particle diameter at the equivalent diameter of a circle of any 50 substrate particles is approximately equal to the average particle diameter at the equivalent diameter of a sphere. In a particle size distribution analyzer, the particle diameter of a single substrate particle is determined as the particle diameter at the equivalent diameter of a sphere. It is preferable to calculate the average particle diameter of the above-mentioned substrate particles using a particle size distribution analyzer.

[0089] (covering layer) The above microcarrier comprises a base particle and a coating layer that covers the outer surface of the base particle. The coating layer is composed of components different from those of the base particle. Examples of components constituting the coating layer include peptides and synthetic resins. The coating layer preferably contains a synthetic resin. The synthetic resin may be used alone or in combination of two or more types.

[0090] From the viewpoint of improving adhesion between microcarriers and cells and maintaining a low degree of swelling of microcarriers, the synthetic resin preferably has a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton. In this case, the synthetic resin may have a polyvinyl alcohol derivative skeleton, or a poly(meth)acrylic acid ester skeleton, or it may have both a polyvinyl alcohol derivative skeleton and a poly(meth)acrylic acid ester skeleton.

[0091] From the viewpoint of enhancing adhesion between microcarriers and cells, the coating layer preferably includes a peptide portion, and more preferably includes a synthetic resin having a peptide portion (peptide skeleton). That is, from the viewpoint of enhancing adhesion between microcarriers and cells, it is more preferable that the synthetic resin has a peptide portion. Note that the embodiment in which the coating layer includes not only embodiments in which the coating layer includes a synthetic resin having a peptide portion, but also embodiments in which the coating layer includes only peptides. From the viewpoint of further enhancing adhesion between microcarriers and cells, it is preferable that the synthetic resin has a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton and a peptide portion.

[0092] In this specification, a "resin having a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton and a peptide portion" may be described as a "peptide-conjugated resin." The above peptide-conjugated resin may have a polyvinyl alcohol derivative skeleton and a peptide portion, a poly(meth)acrylic acid ester skeleton and a peptide portion, or a polyvinyl alcohol derivative skeleton, a poly(meth)acrylic acid ester skeleton and a peptide portion.

[0093] In the peptide-containing resin having the polyvinyl alcohol derivative skeleton described above, it is preferable that the polyvinyl alcohol derivative skeleton and the peptide portion are linked via a linker portion. Therefore, it is preferable that the peptide-containing resin having the polyvinyl alcohol derivative skeleton has a polyvinyl alcohol derivative skeleton, a peptide portion, and a linker portion.

[0094] In the peptide-containing resin having the poly(meth)acrylic acid ester skeleton described above, the poly(meth)acrylic acid ester skeleton and the peptide portion may be bonded via a linker portion, or they may be directly bonded without a linker portion. The peptide-containing resin having the poly(meth)acrylic acid ester skeleton described above may have a poly(meth)acrylic acid ester skeleton, a peptide portion, and a linker portion.

[0095] <Polyvinyl alcohol derivative skeleton> The polyvinyl alcohol derivative skeleton described above is a skeletal portion derived from a polyvinyl alcohol derivative. The polyvinyl alcohol derivative is a compound derived from polyvinyl alcohol. From the viewpoint of further improving the adhesion between microcarriers and cells, the polyvinyl alcohol derivative is preferably a polyvinyl acetal resin, and the polyvinyl alcohol derivative skeleton is preferably a polyvinyl acetal skeleton. That is, the synthetic resin preferably has a polyvinyl acetal skeleton and the peptide portion described above. Only one of the polyvinyl alcohol derivative and the polyvinyl acetal resin may be used, or two or more may be used in combination.

[0096] The polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton preferably have an acetal group, a hydroxyl group, and an acetyl group in their side chains. However, the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton may not have acetyl groups, for example. For example, all of the acetyl groups of the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton may be bonded to the linker, so the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton may not have acetyl groups.

[0097] Polyvinyl acetal resin can be synthesized by acetalizing polyvinyl alcohol with an aldehyde.

[0098] The aldehyde used in the acetalization of polyvinyl alcohol is not particularly limited. Examples of the aldehyde include aldehydes having 1 to 10 carbon atoms. The aldehyde may or may not have a linear aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a linear aldehyde or a cyclic aldehyde. Only one aldehyde may be used, or two or more may be used in combination.

[0099] From the viewpoint of further enhancing the adhesion between microcarriers and cells, the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or pentanal, and more preferably butyraldehyde. Therefore, the polyvinyl acetal resin is more preferably polyvinyl butyral resin, the polyvinyl acetal skeleton is more preferably polyvinyl butyral skeleton, and the synthetic resin is more preferably having a polyvinyl butyral skeleton.

[0100] In the above synthetic resin, the degree of acetalization of the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton (the degree of butyralization in the case of polyvinyl butyral resin) is preferably 40 mol% or more, more preferably 50 mol% or more, more preferably 90 mol% or less, and more preferably 85 mol% or less. If the degree of acetalization is above the lower limit, the cell adhesion can be further improved, and cells can proliferate efficiently. If the degree of acetalization is below the upper limit, the solubility in solvents can be improved.

[0101] In the above-mentioned synthetic resin, the hydroxyl group content (amount of hydroxyl groups) of the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton is preferably 15 mol% or more, more preferably 20 mol% or more, preferably 45 mol% or less, more preferably 30 mol% or less, and even more preferably 25 mol% or less.

[0102] In the above-mentioned synthetic resin, the degree of acetylation (amount of acetyl groups) of the polyvinyl alcohol derivative skeleton and the polyvinyl acetal skeleton is preferably 1 mol% or more, more preferably 2 mol% or more, more preferably 5 mol% or less, and more preferably 4 mol% or less. When the degree of acetylation is above the lower limit and below the upper limit, the reaction efficiency between the polyvinyl acetal resin and the linker can be increased.

[0103] The degree of acetalization, degree of acetylation, and hydroxyl groups of the above polyvinyl alcohol derivative skeleton and the above polyvinyl acetal skeleton are as follows:1 It can be measured using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0104] <Poly(meth)acrylic acid ester skeleton> The above poly(meth)acrylic acid ester skeleton is a skeleton portion derived from poly(meth)acrylic acid ester. The above poly(meth)acrylic acid ester is obtained by polymerization of (meth)acrylic acid ester. The above poly(meth)acrylic acid ester skeleton has a skeleton derived from (meth)acrylic acid ester. The above poly(meth)acrylic acid ester may be used alone, or two or more may be used in combination.

[0105] Examples of the above (meth)acrylic acid esters include alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, polyethylene glycol (meth)acrylates, phosphorylcholine (meth)acrylates, etc. The above (meth)acrylic acid esters may be used individually or in combination of two or more.

[0106] Examples of the alkyl (meth)acrylates mentioned above include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, t-butyl (meth)acrylate, n-octyl (meth)acrylate, isooctyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, isononyl (meth)acrylate, decyl (meth)acrylate, isodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, and isotetradecyl (meth)acrylate.

[0107] The above alkyl (meth)acrylate may be substituted with substituents such as alkoxy groups having 1 to 3 carbon atoms and tetrahydrofurfuryl groups. Examples of such alkyl (meth)acrylates include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0108] Examples of the above-mentioned cyclic alkyl (meth)acrylates include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0109] Examples of the above-mentioned aryl (meth)acrylates include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0110] Examples of the above-mentioned (meth)acrylic polyethylene glycols include methoxy-polyethylene glycol (meth)acrylate, ethoxy-polyethylene glycol (meth)acrylate, hydroxy-polyethylene glycol (meth)acrylate, methoxy-diethylene glycol (meth)acrylate, ethoxy-diethylene glycol (meth)acrylate, hydroxy-diethylene glycol (meth)acrylate, methoxy-triethylene glycol (meth)acrylate, ethoxy-triethylene glycol (meth)acrylate, and hydroxy-triethylene glycol (meth)acrylate.

[0111] Examples of the above-mentioned (meth)acrylic acid phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0112] The above synthetic resin preferably has structural units derived from a (meth)acrylate compound (A) represented by the following formula (A1) or (A2). The above poly(meth)acrylic acid ester skeleton preferably has structural units derived from a (meth)acrylate compound (A) represented by the following formula (A1) or (A2). This makes the hydrophobicity of the coating layer greater, and therefore the water absorption rate of the microcarriers even smaller. As a result, the variation in the initial establishment rate after cell seeding can be reduced, and cells become less likely to detach from the microcarriers in the culture medium. The above (meth)acrylate compound (A) may contain a (meth)acrylate compound represented by the following formula (A1), or it may contain a (meth)acrylate compound represented by the following formula (A2), or it may contain both a (meth)acrylate compound represented by the following formula (A1) and a (meth)acrylate compound represented by the following formula (A2). When the above (meth)acrylate compound (A) includes both the (meth)acrylate compound represented by the following formula (A1) and the (meth)acrylate compound represented by the following formula (A2), R in the following formula (A1) and R in the following formula (A2) may be the same or different. The above (meth)acrylate compound (A) may be used alone or in combination of two or more types. Furthermore, the (meth)acrylate compound represented by the following formula (A1) and the (meth)acrylate compound represented by the following formula (A2) may each be used alone or in combination of two or more types.

[0113] [ka]

[0114] In the above formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0115] [ka]

[0116] In the above formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0117] R in formula (A1) and R in formula (A2) above may each be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. From the viewpoint of improving the solubility of the synthetic resin, it is preferable that R in formula (A1) and R in formula (A2) above are aliphatic hydrocarbon groups. The aliphatic hydrocarbon group may be linear, branched, have a double bond, or not have a double bond. R in formula (A1) and R in formula (A2) above may each be an alkyl group or an alkylene group.

[0118] The number of carbon atoms in R in formula (A1) and formula (A2) is preferably 4 or more, more preferably 6 or more, even more preferably 8 or more, particularly preferably 10 or more, preferably 16 or less, more preferably 14 or less, and most preferably 12. When the number of carbon atoms is above the lower limit, the hydrophobicity of the synthetic resin can be further increased, and therefore the water absorption rate of the microcarriers can be further reduced. When the number of carbon atoms is below the upper limit, the coatability when placing the coating layer material on the surface of the substrate particles can be improved. In particular, when the number of carbon atoms is 12, the water absorption rate of the microcarriers can be reduced even further, and the coatability can be further improved.

[0119] The alkyl (meth)acrylate ester mentioned above is preferably the (meth)acrylate compound (A) mentioned above.

[0120] The synthetic resin having the poly(meth)acrylic acid ester skeleton described above may also have a skeleton derived from monomers other than (meth)acrylic acid esters.

[0121] Examples of monomers other than the (meth)acrylic acid esters mentioned above include (meth)acrylamides and vinyl compounds. Only one of these monomers may be used, or two or more may be used in combination.

[0122] Examples of the above-mentioned (meth)acrylamides include (meth)acrylamide, N-isopropyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, N,N'-dimethyl(meth)acrylamide, (3-(meth)acrylamidopropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, 3-(meth)acryloyl-2-oxazolidinone, N-[3-(dimethylamino)propyl](meth)acrylamide, N-(2-hydroxyethyl)(meth)acrylamide, N-methylol(meth)acrylamide, and 6-(meth)acrylamidohexanoic acid.

[0123] Examples of the vinyl compounds mentioned above include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, and vinylamine.

[0124] <Peptide Division> The peptide portion described above is a structural part derived from a peptide. The peptide portion has an amino acid sequence. The peptide constituting the peptide portion may be an oligopeptide or a polypeptide. Only one type of peptide may be used, or two or more types may be used in combination.

[0125] The number of amino acid residues in the peptide portion is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, preferably 10 or fewer, more preferably 8 or fewer, and even more preferably 6 or fewer. When the number of amino acid residues is above the lower limit and below the upper limit, adhesion to cells after seeding can be further enhanced, and the cell proliferation rate can be further enhanced. However, the number of amino acid residues in the peptide portion may exceed 10 or exceed 15.

[0126] The peptide portion described above preferably has a cell-adherent amino acid sequence. A cell-adherent amino acid sequence refers to an amino acid sequence whose cell-adherent activity has been confirmed by phage display, Sepharose bead method, or plate-coating method. For example, the phage display method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used. For example, the Sepharose bead method described in "Proteins, Nucleic Acids, Enzymes Vol. 45 No. 15 (2000) 2477" can be used. For example, the plate-coating method described in "Proteins, Nucleic Acids, Enzymes Vol. 45 No. 15 (2000) 2477" can be used.

[0127] Examples of the above-mentioned cell-adhering amino acid sequences include RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and TDE sequence (Thr-Asp-Glu). Furthermore, examples of the above-mentioned cell-adherent amino acid sequences include those described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Health Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The above-mentioned peptide portion may have only one of the above-mentioned cell-adherent amino acid sequences, or it may have two or more.

[0128] The above-mentioned cell-adherent amino acid sequence preferably has at least one of the above-mentioned cell-adherent amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, and even more preferably has at least an RGD sequence represented by the following formula (1). In this case, adhesion to cells after seeding can be further enhanced, and the cell proliferation rate can be further increased.

[0129] Arg-Gly-Asp-X...Formula (1)

[0130] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0131] The peptide portion may be linear or may have a cyclic peptide skeleton. From the viewpoint of further enhancing cell proliferation, it is preferable that the peptide portion has a cyclic peptide skeleton. The cyclic peptide skeleton is a cyclic skeleton composed of multiple amino acids. From the viewpoint of more effectively exhibiting the effects of the present invention, it is preferable that the cyclic peptide skeleton is composed of 4 or more amino acids, more preferably of 5 or more amino acids, and preferably of 10 or fewer amino acids.

[0132] In the peptide-containing resin described above, the content of the peptide portion is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, particularly preferably 10 mol% or more, preferably 60 mol% or less, more preferably 50 mol% or less, even more preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide portion is above the lower limit, the adhesion to cells after seeding can be further enhanced, and the cell proliferation rate can be further enhanced. Furthermore, when the content of the peptide portion is below the upper limit, manufacturing costs can be reduced. Note that the content of the peptide portion (mol%) is the amount of substance of the peptide portion relative to the sum of the amounts of substance of each structural unit constituting the peptide-containing resin.

[0133] The content of the peptide portion can be measured, for example, by NMR, FT-IR, or LC-MS.

[0134] <Linker section> The linker portion described above is a structural part derived from the linker. The linker portion is usually located between the polyvinyl alcohol derivative skeleton or the poly(meth)acrylic acid ester skeleton and the peptide portion. The polyvinyl alcohol derivative skeleton or the poly(meth)acrylic acid ester skeleton and the peptide portion are linked via the linker portion. The linker portion is formed by a linker (crosslinking agent). Only one type of linker may be used, or two or more types may be used in combination.

[0135] The linker is preferably a compound having a functional group capable of binding to the peptide, and more preferably a compound having a functional group capable of condensing with the carboxyl group or amino group of the peptide.

[0136] Functional groups that can condense with the carboxyl group or amino group of the above peptide include carboxyl groups, thiol groups, amino groups, hydroxyl groups, and cyano groups.

[0137] From the viewpoint of reacting well with peptides, the linker is preferably a compound having a carboxyl group or an amino group, and more preferably a compound having a carboxyl group.

[0138] When obtaining a peptide-containing resin having a polyvinyl alcohol derivative skeleton, examples of linkers having carboxyl groups include (meth)acrylic acid and carboxyl-containing acrylamide. By using a polymerizable unsaturated carboxylic acid (carboxylic acid monomer) as the linker having carboxyl groups, the carboxylic acid monomer can be polymerized by graft polymerization when the linker is introduced, thereby increasing the number of carboxyl groups that can react with the peptide.

[0139] From the viewpoint of effectively bonding the polyvinyl alcohol derivative and the peptide, the linker is preferably (meth)acrylic acid, and more preferably acrylic acid.

[0140] When obtaining a peptide-containing resin having a poly(meth)acrylic acid ester skeleton, it is preferable that the linker has a functional group capable of binding to the (meth)acrylic acid ester. Examples of functional groups capable of binding to the (meth)acrylic acid ester include vinyl groups, (meth)acryloyl groups, and allyl groups. It is more preferable that the linker has a (meth)acryloyl group as a functional group capable of binding to the (meth)acrylic acid ester, and is preferably a compound having a carboxyl group or an amino group and a (meth)acryloyl group.

[0141] Examples of linkers used to obtain peptide-containing resins having a poly(meth)acrylic acid ester skeleton include (meth)acrylic acid, itaconic acid, and acrylamide.

[0142] From the viewpoint of effectively bonding poly(meth)acrylic acid esters and peptides, the linker is preferably (meth)acrylic acid or itaconic acid, and more preferably (meth)acrylic acid.

[0143] <Further details about the coating layer> The weight-average molecular weight of the above synthetic resin is preferably 10,000 or more, more preferably 50,000 or more, preferably 1,200,000 or less, and more preferably 600,000 or less. When the weight-average molecular weight is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively. When the weight-average molecular weight is below the upper limit, the spreadability of cells during cell culture can be enhanced even more effectively.

[0144] The weight-average molecular weight can be measured, for example, by the following method. Dissolve the synthetic resin in tetrahydrofuran (THF) to prepare a 0.2 wt% solution of the synthetic resin. Next, evaluate the solution using a gel permeation chromatography (GPC) analyzer (APC system, Waters) under the following measurement conditions.

[0145] Column: HSPgel HR MB-M 6.0×150mm Flow rate: 0.5mL / min Column temperature: 40℃ Injection volume: 10μL Detectors: RI, PDA Standard sample: Polystyrene

[0146] The coating layer may contain only a resin having the polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton. The coating layer may also contain a resin having the polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton but without a peptide portion, and a peptide-containing resin. The coating layer may also contain other components, such as a resin that does not have either the polyvinyl alcohol derivative skeleton or the poly(meth)acrylic acid ester skeleton. Examples of these other components include polyolefin resins, polyether resins, polyesters, epoxy resins, polyamide resins, polyimide resins, polyurethane resins, polycarbonate resins, cellulose, and polypeptides. Only one of these other components may be used, or two or more may be used in combination.

[0147] The coating layer may have only a layer containing a resin having the polyvinyl alcohol derivative skeleton or poly(meth)acrylic acid ester skeleton (hereinafter sometimes referred to as "layer X"). The coating layer may also have a layer that does not contain a resin having the polyvinyl alcohol derivative skeleton or poly(meth)acrylic acid ester skeleton (hereinafter sometimes referred to as "layer Y"). The coating layer may have both layer X and layer Y. When the coating layer has both layer X and layer Y, it is preferable that layer Y is located on the substrate particle side and layer X is located outside layer Y. In this case, the adhesion between the microcarrier and the cells can be further enhanced.

[0148] The peptide-containing resin is preferably present at least on the outer surface of the microcarrier. The outermost layer of the microcarrier is preferably a layer containing the peptide-containing resin. In this case, the adhesion between the microcarrier and the cell can be further enhanced.

[0149] The content of the synthetic resin in 100% by weight of the above coating layer is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). When the content of the synthetic resin is above the lower limit, the effects of the present invention can be exhibited even more effectively. The content of the synthetic resin in 100% by weight of the above coating layer may be 100% by weight or less, or less than 100% by weight.

[0150] In 100% by weight of the above coating layer, the content of the resin having the polyvinyl alcohol derivative skeleton or poly(meth)acrylic acid ester skeleton or the peptide-containing resin is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). If the content of the resin having the polyvinyl alcohol derivative skeleton or poly(meth)acrylic acid ester skeleton or the peptide-containing resin is above the lower limit, the effects of the present invention can be exhibited even more effectively. In 100% by weight of the above coating layer, the content of the resin having the polyvinyl alcohol derivative skeleton or poly(meth)acrylic acid ester skeleton or the peptide-containing resin may be 100% by weight or less, or less than 100% by weight.

[0151] Of the total surface area of ​​the above-mentioned substrate particles, the surface area covered by the above-mentioned coating layer (coverage rate) is preferably 50% or more, more preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, particularly preferably 99% or more, and most preferably 100%. When the above-mentioned coverage rate is above the above lower limit, the adhesion between the microcarrier and the cell can be further enhanced, and the effects of the present invention can be exhibited more effectively. The above-mentioned coverage rate may be 100% or less, less than 100%, or 99% or less.

[0152] The above coverage rate is determined by observing the microcarriers with an electron microscope or optical microscope and calculating the percentage of the surface area covered by the coating layer relative to the projected area of ​​the substrate particles.

[0153] The thickness of the coating layer is preferably 10 nm or more, more preferably 50 nm or more, more preferably 1000 nm or less, and more preferably 500 nm or less. When the thickness of the coating layer is above the lower limit and below the upper limit, the adhesion between the microcarrier and the cell can be further enhanced. Furthermore, when the thickness of the coating layer is above the lower limit and below the upper limit, the effects of the present invention can be exhibited even more effectively.

[0154] The thickness of the coating layer can be measured, for example, by observing the cross-section of the microcarrier using a scanning electron microscope (SEM). Preferably, the thickness of the coating layer is calculated by averaging the thickness of five arbitrary coating layers, and more preferably by averaging the thickness of the entire coating layer. The thickness of the coating layer is preferably determined by calculating the average thickness of the coating layer for 50 arbitrary microcarriers.

[0155] Examples of methods for obtaining a peptide-containing resin having the polyvinyl alcohol derivative skeleton described above include the following methods.

[0156] A polyvinyl alcohol derivative (e.g., polyvinyl acetal resin) is reacted with a linker to obtain a reaction product in which the polyvinyl acetal resin and the linker are bonded. The obtained reaction product is then reacted with a peptide to obtain a peptide-containing resin having a polyvinyl alcohol derivative skeleton (polyvinyl acetal skeleton).

[0157] Examples of methods for obtaining a peptide-containing resin having the poly(meth)acrylic acid ester skeleton described above include the following methods.

[0158] An acrylic resin is obtained by polymerizing monomers containing (meth)acrylic acid ester. The obtained acrylic resin is reacted with a peptide and a linker, if necessary, to obtain a peptide-containing resin having a poly(meth)acrylic acid ester skeleton.

[0159] Examples of methods for obtaining a peptide-containing resin having the above-mentioned polyvinyl alcohol derivative skeleton and the above-mentioned poly(meth)acrylic acid ester skeleton include the following methods.

[0160] A resin having a polyvinyl alcohol derivative skeleton and a poly(meth)acrylic acid ester skeleton is obtained by the following methods (i), (ii), or (iii): (i) A polyvinyl acetal resin is synthesized using polyvinyl alcohol copolymerized with an acrylic acid ester. (ii) A polyvinyl acetal resin is synthesized using polyvinyl alcohol and polyvinyl alcohol copolymerized with an acrylic acid ester. (iii) An acrylic acid ester is graft copolymerized onto the polyvinyl acetal resin. The resin obtained by the above methods (i), (ii), or (iii) is reacted with a peptide and a linker used as needed to obtain a peptide-containing resin having the above polyvinyl alcohol derivative skeleton and the above poly(meth)acrylic acid ester skeleton.

[0161] Methods for obtaining microcarriers by arranging the coating layer on the surface of the above-mentioned substrate particles include, for example, the following methods (1) and (2).

[0162] Method (1): The peptide-containing resin obtained by the above method is dissolved in a solvent to obtain a liquid containing the peptide-containing resin. Microcarriers can be prepared by spraying the liquid containing the peptide-containing resin onto substrate particles or by separating substrate particles that have been impregnated with the liquid containing the peptide-containing resin, thereby providing a layer (coating layer) containing the peptide-containing resin on the outer surface of the substrate particles.

[0163] Method (2): A resin having a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton (resin before peptide bonding) is prepared. This resin is dissolved in a solvent to obtain a resin-containing solution. The resin-containing solution is sprayed onto substrate particles, or substrate particles impregnated with the resin-containing solution are separated to obtain particles in which a layer containing a resin having a polyvinyl alcohol derivative or poly(meth)acrylic acid ester is disposed on the outer surface of the substrate particles. The obtained particles are reacted with the resin having a polyvinyl alcohol derivative or poly(meth)acrylic acid ester contained in the layer, a peptide, and a linker used as needed, using the method described above. In this way, microcarriers can be produced on the outer surface of the substrate particles, comprising a layer containing a resin having a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton and no peptide portion, and a layer containing a peptide-containing resin, as a coating layer.

[0164] (More details about microcarriers) The above microcarriers are used for culturing cells.

[0165] Examples of the cells mentioned above include animal cells from humans, mice, rats, pigs, cattle, and monkeys. Other examples of the cells include somatic cells, such as stem cells, progenitor cells, and mature cells. These somatic cells may also be cancer cells.

[0166] Examples of the above-mentioned stem cells include mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0167] Examples of the mature cells mentioned above include nerve cells, cardiomyocytes, retinal cells, and hepatocytes.

[0168] The above-mentioned microcarriers are preferably used for three-dimensional cell culture. Three-dimensional culture is a method of culturing cells with thickness in the vertical direction, in contrast to two-dimensional culture, which involves culturing cells on a flat surface such as a plate.

[0169] The above microcarrier is preferably used in serum-free culture media. The above microcarrier can enhance cell adhesion even in serum-free culture media that do not contain feeder cells or adhesion proteins, and in particular, it can further improve the initial colonization rate after cell seeding. Furthermore, the above microcarrier can exhibit the effects of the present invention even in serum-free culture media. In particular, when the coating layer contains the above peptide-containing resin, the effects of enhancing cell adhesion and improving the initial colonization rate after cell seeding are effectively exhibited even in serum-free culture media.

[0170] Preferably, the above microcarrier contains substantially no animal-derived raw materials. By not containing animal-derived raw materials, it is possible to provide a microcarrier that is highly safe and has less variation in quality during manufacturing. "Substantially no animal-derived raw materials" means that the amount of animal-derived raw materials in the microcarrier is 3% by weight or less. Preferably, the amount of animal-derived raw materials in the above microcarrier is 1% by weight or less, and most preferably 0% by weight. In other words, it is most preferable that the above microcarrier contains no animal-derived raw materials at all.

[0171] (Methods for culturing cells) Cells can be cultured using the above-mentioned microcarriers. The cell culture method according to the present invention is a cell culture method using the above-mentioned microcarriers. Examples of the above-mentioned cells include the cells described above.

[0172] The cell culture method described above preferably includes a step of adhering the cells to the microcarrier. The cells may also be in the form of cell aggregates. These cell aggregates can be obtained by adding a cell detachment agent to a confluent culture vessel and uniformly disrupting the cells by pipetting. The cell detachment agent is not particularly limited, but ethylenediamine / phosphate buffer solution is preferred. The size of the cell aggregates is preferably 50 μm to 200 μm.

[0173] The present invention will be described in more detail below with reference to examples and comparative examples. The present invention is not limited to these examples.

[0174] Furthermore, the structural unit content in the obtained resin was determined after dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide). 1 The results were measured using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0175] (Example 1) (1) Preparation of base particle A 800 parts by weight of divinylbenzene (57% purity) and 200 parts by weight of styrene were mixed to obtain a mixture. 20 parts by weight of benzoyl peroxide were added to the mixture and stirred until uniformly dissolved to obtain a monomer mixture. 4000 parts by weight of a 2% aqueous solution of polyvinyl alcohol with a molecular weight of approximately 1700, dissolved in pure water, was placed in a reaction vessel. Next, the obtained monomer mixture was added to the reaction vessel and stirred for 4 hours to adjust the particle size so that the monomer droplets reached a predetermined particle size. Then, the reaction was carried out under a nitrogen atmosphere at 85°C for 9 hours to perform a polymerization reaction of the monomer droplets and obtain particles. The obtained particles were washed several times with hot water, methanol, and acetone, respectively, and then classified to obtain base particle A with an average particle size of 100 μm and a particle size CV value of 1%. Base particle A is a resin particle of divinylbenzene copolymer (indicated as DVB in the table).

[0176] (2) Preparation of polyvinyl acetal resin In a reactor equipped with a stirring device, 2700 mL of deionized water and 300 parts by weight of polyvinyl alcohol with an average degree of polymerization of 1700 and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst to achieve a hydrochloric acid concentration of 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde was added while stirring. Then, 148 parts by weight of n-butyraldehyde was added to precipitate white particulate polyvinyl acetal resin (polyvinyl butyral resin). Fifteen minutes after precipitation, 35% by weight hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8% by weight, and the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, then the polyvinyl butyral resin was washed with water and dried to obtain polyvinyl acetal resin (polyvinyl butyral resin, average degree of polymerization 1700, degree of acetalization (degree of butyralization) 70 mol%, hydroxyl group content 27 mol%, degree of acetylation 3 mol%).

[0177] (3) Formation of the linker 99 parts by weight of the obtained polyvinyl acetal resin and 1 part by weight of acrylic acid (linker) were dissolved in 300 parts by weight of THF (tetrahydrofuran), and the mixture was reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to form a linker portion by graft copolymerization of the polyvinyl acetal resin and acrylic acid.

[0178] (4) Preparation of polyvinyl acetal resin coated particles with linker portion One part by weight of polyvinyl acetal resin with a linker formed was dissolved in 19 parts by weight of butanol. One part by weight of base particle A was added to the resulting solution and stirred. The solution was then filtered, washed with pure water, and vacuum dried at 60°C for 5 hours to obtain polyvinyl acetal resin coated particles with a linker formed.

[0179] (5) Fabrication of microcarriers A linear peptide (5 amino acid residues) having the amino acid sequence Gly-Arg-Gly-Asp-Ser was prepared. 1 part by weight of this peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (a bonding agent) were added to phosphate-buffered saline that did not contain either calcium or magnesium, so that the final concentration of the peptide was 1 mM, to prepare a peptide-containing solution. 1 part by weight of polyvinyl acetal resin-coated particles with a linker moiety was added to 20 parts by weight of the obtained peptide-containing solution, and the carboxyl group of the linker moiety and the amino group of Gly in the peptide were dehydrated and condensed. The resulting suspension was filtered, washed with pure water, and vacuum-dried at 60°C for 5 hours to obtain microcarriers. In the table, the peptide-containing resin having a polyvinyl alcohol derivative skeleton (polyvinyl acetal skeleton) obtained by the above method is referred to as resin X1. Resin X1 has the amino acid sequence Gly-Arg-Gly-Asp-Ser as the peptide moiety.

[0180] (Example 2) (1) Preparation of base particle B A polymerization reaction was carried out in the same manner as in Example 1 to obtain particles. By performing a classification operation on the obtained particles, base particle B with an average particle size of 200 μm and a particle size CV value of 1% was obtained.

[0181] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that the obtained substrate particles B were used.

[0182] (Example 3) (1) Preparation of base particle C A polymerization reaction was carried out in the same manner as in Example 1 to obtain particles. By performing a classification operation on the obtained particles, base particle C with an average particle diameter of 300 μm and a particle diameter CV value of 1% was obtained.

[0183] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that the obtained substrate particles C were used.

[0184] (Example 4) (1) Preparation of base particle D A polymerization reaction was carried out in the same manner as in Example 1 to obtain particles. By performing a classification operation on the obtained particles, base particle D with an average particle diameter of 400 μm and a particle diameter CV value of 1% was obtained.

[0185] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that the obtained substrate particles D were used.

[0186] (Example 5) (1) Preparation of base particle E Particles were obtained in the same manner as in Example 1, except that the amount of divinylbenzene was changed from 800 parts by weight to 30 parts by weight, and the amount of styrene was changed from 200 parts by weight to 970 parts by weight. By performing a classification operation on the obtained particles, base particle E with an average particle diameter of 300 μm and a particle diameter CV value of 5% was obtained. Base particle E is a resin particle of polystyrene-divinylbenzene copolymer (indicated as PS97% / DVB3% in the table).

[0187] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that the obtained substrate particles E were used.

[0188] (Example 6) Base particle C was used as the base particle.

[0189] (2) Preparation of acrylic resin An acrylic monomer solution was obtained by dissolving 10 parts by weight of dodecyl acrylate and 2.7 parts by weight of acrylic acid in 27 parts by weight of tetrahydrofuran. 0.0575 parts by weight of Irgacure 184 (BASF) was dissolved in the obtained acrylic monomer solution, and the resulting solution was coated onto a PET film. The coated object was exposed to light at a wavelength of 365 nm and an integrated light intensity of 2000 mJ / cm² using a UV conveyor (ECS301G1, iGraphics) at 25°C. 2A (meth)acrylic copolymer solution was obtained by irradiation. The obtained (meth)acrylic copolymer solution was vacuum-dried at 80°C for 3 hours to obtain an acrylic resin having a linker portion.

[0190] (3) Preparation of acrylic resin coated particles One part by weight of the obtained acrylic resin was dissolved in 19 parts by weight of butanol. One part by weight of the base particles was added to this solution and stirred. The solution was then filtered, washed with pure water, and vacuum dried at 60°C for 5 hours to obtain acrylic resin coated particles.

[0191] (4) Fabrication of microcarriers The obtained acrylic resin-coated particles were used. In addition, a cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (5 amino acid residues, with Arg and Lys forming a cyclic skeleton, and Phe being the D-isomer) was prepared as the peptide. Using this peptide, microcarriers were obtained in the same manner as in Example 1, except that the carboxyl group in the structural unit derived from acrylic acid of the acrylic resin and the amino group of Lys in the peptide were subjected to dehydration condensation. In the table, the peptide-containing resin having a poly(meth)acrylic acid ester skeleton obtained by the above method is referred to as resin X2. Resin X2 has the amino acid sequence Arg-Gly-Asp-Phe-Lys (cyclic peptide skeleton) as the peptide portion.

[0192] (Example 7) (1) Preparation of base particle F Micropearl GS-L300 (manufactured by Sekisui Chemical Co., Ltd., average particle size 300 μm, particle size CV value 7%, polyfunctional acrylic resin particles) was prepared. By performing a classification operation on these particles, base particle F with an average particle size of 300 μm and a particle size CV value of 1% was obtained. Base particle F is a resin particle of acrylic resin (indicated as ACR in the table).

[0193] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that the obtained substrate particles F were used.

[0194] (Comparative Example 1) (1) Preparation of base particle G Corning's "Untreated Microcarriers" (base particles are polystyrene particles, indicated as PS in the table) were subjected to a classification process, and the resulting particles with a particle size CV value of 1% were used as base particle G.

[0195] (2) Fabrication of microcarriers Microcarriers were prepared in the same manner as in Example 1, except that base particle G was used.

[0196] (Comparative Example 2) The obtained substrate particles C themselves were used as microcarriers.

[0197] (evaluation) (1) Specific gravity of substrate particles and microcarriers The specific gravity of the obtained substrate particles and microcarriers was measured using a true hydrometer (Shimadzu Corporation's "AccuPic II") in a dry state and under an argon gas atmosphere.

[0198] (2) Average particle diameter of microcarriers and coefficient of variation (CV value) of particle diameter The obtained microcarriers were observed using a scanning electron microscope. The average particle diameter and coefficient of variation (CV) of particle diameter at the equivalent circle diameter were calculated for 50 arbitrary microcarriers.

[0199] (3) Thickness of the coating layer The cross-sections of the obtained microcarriers were observed using a scanning electron microscope. The thickness of the coating layer was measured for each of 50 randomly selected microcarriers, and the average value was defined as the thickness of the microcarrier's coating layer.

[0200] (4) Breaking strength of microcarriers The fracture strength of the obtained microcarriers was measured using the method described above. A Shimadzu Corporation "Microautograph MST-I" was used as the micro-strength evaluation test device. In the table, ">2000" indicates that the microcarriers did not have a fracture strength of 2000 mN or less, meaning the fracture strength of the microcarriers exceeded 2000 mN.

[0201] (5) Load at the inflection point of the compression displacement curve Compression tests were performed on the obtained microcarriers using the method described above. The load value (mN) and compressive displacement (μm) were measured, and a compressive displacement curve showing the relationship between compressive displacement (x axis) and load value (y axis) was created. The inflection point of the obtained compressive displacement curve was determined, and the load at that inflection point was calculated. If the compressive displacement curve did not have an inflection point at loads of 700 mN or less, it was indicated with "-" in the table.

[0202] (6) Water absorption rate of microcarriers The obtained microcarriers were dried in an oven at 100°C for 8 hours. 100.0 mg of these microcarriers were weighed and left for 24 hours in an environment at 37°C and 95% RH relative humidity. The weight of the microcarriers after standing was measured, and the water absorption rate of the microcarriers was calculated using the following formula.

[0203] Water absorption rate (weight %)=(W2-W1) / W1×100 W1: Weight of microcarriers before standing (mg) W2: Weight of microcarriers after standing (mg)

[0204] (7) Evaluation of cell culture (cell count) A 12-well plate (Corning, flat-bottom, untreated) was used as the culture plate. The following liquid culture mediums and ROCK (Rho-binding kinase) specific inhibitors were also prepared.

[0205] STEMFIT culture medium (manufactured by Ajinomoto Healthy Supply Co., Ltd.) ROCK-Inhibitor (Y27632)

[0206] Place 3.8 × 10¹ confluent h-iPS cells 201B7 in a φ35mm dish. 4 The cells were added to 1 mL of 0.5 mM ethylenediaminetetraacetic acid / phosphate buffer solution and allowed to stand at room temperature for 5 minutes. After removing the ethylenediaminetetraacetic acid / phosphate buffer solution, a cell suspension was obtained by pipetting with 1 mL of liquid medium. The resulting microcarrier (60 cm² in surface area) 2 The entire amount of the obtained cell suspension was added to a culture plate containing 4 mL of liquid medium. This culture plate was placed in a shaker and cultured with shaking at 37°C, CO2 concentration 5%, and 46 rpm.

[0207] Three days after the start of shaking culture, 4 ml of supernatant liquid medium was replaced with 4 ml of fresh liquid medium. Five days after the start of shaking culture, 4 ml of supernatant liquid medium was removed from the culture plate. Next, 1.0 ml of TryPLE Express detachment solution was added to the culture plate and suspended by pipetting. The suspension, along with the microcarriers, was added to a cell strainer to separate the microcarriers from the cell suspension. The number of cells in the resulting cell suspension was determined using a cell counter (Chemometec "NucleoCounter NC-3000").

[0208] <Criteria for evaluating cell culture> AA: Cell count is 1.6 × 10⁻⁶ 5 pcs or more A: Cell count is 1.2 × 10 5 pcs or more, 1.6×10 5 Less than one B: Cell count is 3.8 × 10 4 pcs or more, 1.2×10 5 Less than one C: Cell count is 3.8 × 10 4 Less than one

[0209] (8) Microcarrier damage The microcarriers after the evaluation described in "(7) Cell Culture Evaluation" above were observed using a phase-contrast microscope to check whether or not damage had occurred to the microcarriers. In the table, "Present" is written when damaged microcarriers were observed, and "Absent" is written when damaged microcarriers were not observed. In Comparative Example 1, damaged microcarriers were observed as shown in Figure 2.

[0210] Details and results are shown in Tables 1 and 2 below.

[0211] [Table 1]

[0212] [Table 2] [Explanation of symbols]

[0213] 1…Microcarriers for cell culture 2...Base material particles 3…Covering layer

Claims

1. Substrate particles and The system comprises a coating layer that covers the outer surface of the substrate particles, A microcarrier for cell culture with a breaking strength of 1000 mN or more.

2. The microcarrier for cell culture according to claim 1, wherein the compression displacement curve obtained when a compression test is performed does not have an inflection point when the load is 700 mN or less.

3. A microcarrier for cell culture according to claim 1 or 2, wherein the water absorption rate is 10% by weight or less.

4. The microcarrier for cell culture according to any one of claims 1 to 3, wherein the coating layer comprises a synthetic resin.

5. The microcarrier for cell culture according to claim 4, wherein the synthetic resin has a polyvinyl alcohol derivative skeleton or a poly(meth)acrylic acid ester skeleton.

6. The microcarrier for cell culture according to any one of claims 1 to 5, wherein the coating layer includes a peptide portion.

7. The microcarrier for cell culture according to any one of claims 1 to 6, wherein the base material particles are resin particles.

8. The microcarrier for cell culture according to any one of claims 1 to 7, wherein the base particles include a polymer of monomers having ethylenically unsaturated groups.

9. The microcarrier for cell culture according to claim 8, wherein the polymer of the monomer having an ethylenically unsaturated group is an acrylic resin, a divinylbenzene polymer, or a divinylbenzene copolymer.

10. Specific gravity is 1.0 g / cm³ ３ 2.0g / cm or more ３ The following is a microcarrier for cell culture according to any one of claims 1 to 9.

11. A microcarrier for cell culture according to any one of claims 1 to 10, wherein the average particle size is 100 μm or more and 1500 μm or less.

12. A microcarrier for cell culture according to any one of claims 1 to 11, wherein the CV value of the particle size is 10% or less.

13. A method for culturing cells, comprising the step of adhering cells to a cell culture microcarrier according to any one of claims 1 to 12.

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