Scaffold material for cell culture

Abstract

Provided is a scaffold material for cell culture, with which cell culture stability can be maintained over a long period of time.　This scaffold material for cell culture contains a peptide-containing (meth)acrylic copolymer having: a (meth)acrylic copolymer part; and a peptide part bonded to the (meth)acrylic copolymer part. If the peptide-containing (meth)acrylic copolymer is subjected to high performance liquid chromatography measurements under condition 1 below, a peak top of a main peak having the largest area among signals is not detected at a residence time of 4 minutes or less.　[Condition 1] Column: C18 (internal diameter 3.0 mm × length 150 mm, particle diameter of packing material 3.5μm). Column temperature: 40ºC. Flow rate: 0.3 mL / min. Detector: evaporative light scattering detector. Mobile phase A (liquid A): 0.1 wt% aqueous solution of formic acid. Mobile phase B (liquid B): isopropyl alcohol. Residence time: 0 min. Liquid A / liquid B (volume ratio): 70% / 30%. Residence time 0-15 min: liquid A / liquid B (volume ratio) changes from 70% / 30% to 0% / 100%. Residence time 15-30 min: liquid A / liquid B (volume ratio) maintained at 0% / 100%

Description

Scaffold materials for cell culture

[0001] The present invention relates to a scaffold material for cell culture.

[0002] Animal cells from humans, mice, rats, pigs, cattle, monkeys, etc. are used in research and development in academic fields, drug discovery, regenerative medicine, etc. Scaffold materials used to culture animal cells include adhesive proteins such as laminin and vitronectin, and natural polymeric materials such as Matrigel derived from mouse sarcoma.

[0003] Also known are scaffold materials using synthetic resins and scaffold materials using synthetic resins to which peptides are bound.

[0004] Patent Document 1 below discloses a cell culture article coated with a composition containing a polymer in which an acrylic polymer and a polypeptide are bound. In Patent Document 1, a hydrophilic acrylic polymer obtained by polymerizing a hydrophilic acrylic monomer is used as the acrylic polymer.

[0005] Patent Document 2 below discloses an adhesive cell culture coating composition in which a water-insoluble polymer compound is dissolved in a lower alcohol or a mixed solvent of a lower alcohol and water. Patent Document 2 describes a copolymer of a (meth)acrylic acid derivative chemically modified with a peptide and a hydrophilic acrylate compound as the water-insoluble polymer compound. Furthermore, the copolymer used has a relatively high content of the hydrophilic acrylate compound.

[0006] WO2012 / 158235A2 Japanese Patent Application Publication No. 5-292957

[0007] Scaffold materials for cell culture using acrylic copolymers to which peptides are bound are known, as described in Patent Documents 1 and 2. Cells can be cultured in a liquid medium using scaffold materials that have been processed or molded into a predetermined shape.

[0008] However, conventional scaffold materials such as those described in Patent Documents 1 and 2 use acrylic copolymers that are relatively hydrophilic, and therefore the scaffold material is prone to gradually eluting into the liquid medium during cell culture or peeling off from the container during cell culture. Therefore, with conventional scaffold materials such as those described in Patent Documents 1 and 2, the cell growth rate decreases as the number of days of cell culture increases, and the cell culture stability is prone to decrease.

[0009] An object of the present invention is to provide a scaffold material for cell culture that can maintain the stability of cell culture for a long period of time.

[0010] The scaffold material for cell culture according to the first invention of the present application comprises a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bound to the (meth)acrylic copolymer portion, and when the peptide-containing (meth)acrylic copolymer is subjected to high performance liquid chromatography measurement under the following condition 1, the peak top of the main peak having the largest area among the signals is not detected within a retention time of 4 minutes.

[0011] [Condition 1] Column: C18 (inner diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3 mL / min Detector: evaporative light scattering detector Mobile phase A (liquid A): 0.1 wt% formic acid aqueous solution Mobile phase B (liquid B): isopropyl alcohol Retention time 0 min: liquid A / liquid B (volume ratio) = 70% / 30% Retention time 0 min to 15 min: liquid A / liquid B (volume ratio) = changed from 70% / 30% to 0% / 100% Retention time 15 min to 30 min: liquid A / liquid B (volume ratio) = maintained at 0% / 100%

[0012] In a specific aspect of the cell culture scaffold material according to the first invention of the present application, when the peptide-containing (meth)acrylic copolymer is subjected to high performance liquid chromatography measurement under the condition 1, the retention time of the peak top of the main peak having the largest area among the detected signals is 5 minutes or more and 30 minutes or less.

[0013] A scaffold material for cell culture according to a second aspect of the present invention comprises a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bound to the (meth)acrylic copolymer portion, and when the peptide-containing (meth)acrylic copolymer is subjected to high performance liquid chromatography measurement under the following condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals is 5 minutes or more and 30 minutes or less.

[0014] [Condition 2] Column: C18 (inner diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3 mL / min Detector: evaporative light scattering detector Mobile phase A (liquid A): 0.1 wt / vol formic acid methanol Mobile phase B (liquid B): THF / isopropyl alcohol = 7 / 3 (volume ratio) Retention time 0 to 2 min: Liquid A / Liquid B (volume ratio) = 100% / 0% maintained Retention time 2 to 5 min: Liquid A / Liquid B (volume ratio) = Changed from 100% / 0% to 90% / 10% Retention time 5 to 17 min: Liquid A / Liquid B (volume ratio) = Changed from 90% / 10% to 0% / 100% Retention time 17 to 30 min: Liquid A / Liquid B (volume ratio) = 0% / 100% maintained

[0015] Hereinafter, the first and second inventions of the present application may be collectively referred to as the present invention.

[0016] In a specific aspect of the scaffold material for cell culture according to the present invention, the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or the following formula (A2):

[0017]

[0018] In the formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0019]

[0020] In the formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0021] In a specific aspect of the scaffold material for cell culture according to the present invention, the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (B) having a functional group capable of reacting with an amino group or a carboxyl group, and in the peptide-containing (meth)acrylic copolymer, the peptide portion is bonded to the functional group capable of reacting with the amino group or the carboxyl group.

[0022] In a specific aspect of the scaffold material for cell culture according to the present invention, the content of structural units derived from the (meth)acrylate compound (A) is 25 mol % or more and 98 mol % or less, based on 100 mol % of all structural units in the (meth)acrylic copolymer portion.

[0023] In a specific aspect of the scaffold material for cell culture according to the present invention, the peptide-containing (meth)acrylic copolymer has a number average molecular weight of 5,000 or more.

[0024] In a specific aspect of the scaffold material for cell culture according to the present invention, the peptide content in the peptide-containing (meth)acrylic copolymer is 0.5 mol % or more and 25 mol % or less.

[0025] In a specific aspect of the scaffold material for cell culture according to the present invention, the peptide portion has an RGD sequence.

[0026] According to the present invention, it is possible to provide a scaffold material for cell culture that can maintain the stability of cell culture for a long period of time.

[0027] Fig. 1 is a cross-sectional view schematically showing a cell culture vessel according to one embodiment of the present invention. Fig. 2 is a cross-sectional view schematically showing a cell culture microcarrier according to one embodiment of the present invention. Fig. 3 is a phase-contrast microscope photograph taken 24 hours, 48 ​​hours, and 72 hours after cell seeding when cell culture was performed using the scaffold materials for cell culture obtained in Examples 1 and 8 and Comparative Example 2.

[0028] The present invention will be described in detail below. In this specification, the term "scaffold material for cell culture" may be abbreviated as "scaffold material".

[0029] A scaffold material according to a first aspect of the present invention includes a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bound to the (meth)acrylic copolymer portion. When the peptide-containing (meth)acrylic copolymer is subjected to high-performance liquid chromatography measurement under the following condition 1, the peak top of the main peak having the largest area among the signals is not detected within a retention time of 4 minutes.

[0030] [Condition 1] Column: C18 (inner diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3 mL / min Detector: evaporative light scattering detector Mobile phase A (liquid A): 0.1 wt% formic acid aqueous solution Mobile phase B (liquid B): isopropyl alcohol Retention time 0 min: liquid A / liquid B (volume ratio) = 70% / 30% Retention time 0 min to 15 min: liquid A / liquid B (volume ratio) = changed from 70% / 30% to 0% / 100% Retention time 15 min to 30 min: liquid A / liquid B (volume ratio) = maintained at 0% / 100%

[0031] Specifically, the high performance liquid chromatography measurement under condition 1 (hereinafter, "high performance liquid chromatography" may be abbreviated as "HPLC") can be carried out, for example, as follows.

[0032] First, a measurement sample can be obtained by adjusting the peptide-containing (meth)acrylic copolymer to a concentration of 2 mg / mL using a mixed solution of 0.1 wt % aqueous formic acid and isopropyl alcohol (0.1 wt % aqueous formic acid / isopropyl alcohol = 7 / 3 [volume ratio]) as a solvent.

[0033] When the scaffold material contains components other than the peptide-containing (meth)acrylic copolymer, the isolated peptide-containing (meth)acrylic copolymer is used as the measurement sample. When the scaffold material is in a state of being coated on a culture vessel or the like, the scaffold material coated on the culture vessel or the like is immersed in ethanol, and the peptide-containing (meth)acrylic copolymer dissolved in the ethanol is used as the measurement sample.

[0034] Examples of measuring instruments that can be used include an HPLC device (Shimadzu Corporation, High-Pressure Gradient HPLC System Prominence), an HPLC column (Waters Corporation, XBridge (registered trademark) C18 [internal diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm]), and an evaporative light scattering detector (Shimadzu Corporation, ELSD_LTII). The analysis can be performed, for example, by the following procedure.

[0035] A 0.1 wt % formic acid aqueous solution is used as mobile phase A (liquid A), and isopropyl alcohol is used as mobile phase B (liquid B). At the start of measurement, the HPLC apparatus is filled with a mixed solvent of mobile phase A / mobile phase B in a volume ratio of 7 / 3. In this state, the measurement sample (injection volume: 20 μL) is injected. Then, immediately after sample injection, the proportion of mobile phase B in the mobile phase is increased at a constant rate over 15 minutes so that the proportion of mobile phase B in the mobile phase reaches 100% by volume. After 15 minutes (at this point, the mobile phase is completely replaced with mobile phase B), mobile phase B is allowed to flow for 15 minutes. The column temperature is 40°C, and the total flow rate is 0.3 mL / min.

[0036] Nitrogen gas is used as the nebulizer gas for the evaporative light scattering detector. The gas supply pressure is 350 kPa, and the drift tube temperature is 40 ° C. The baseline can be determined by analyzing a blank test solution prepared in the same manner as the preparation of the analytical sample, except that the scaffold material is dissolved.

[0037] A scaffold material according to a second aspect of the present invention includes a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bound to the (meth)acrylic copolymer portion. When the peptide-containing (meth)acrylic copolymer is subjected to high-performance liquid chromatography under the following condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals is 5 minutes or more and 30 minutes or less.

[0038] [Condition 2] Column: C18 (inner diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3 mL / min Detector: evaporative light scattering detector Mobile phase A (liquid A): 0.1 wt / vol formic acid methanol Mobile phase B (liquid B): THF / isopropyl alcohol = 7 / 3 (volume ratio) Retention time 0 to 2 min: Liquid A / Liquid B (volume ratio) = 100% / 0% maintained Retention time 2 to 5 min: Liquid A / Liquid B (volume ratio) = Changed from 100% / 0% to 90% / 10% Retention time 5 to 17 min: Liquid A / Liquid B (volume ratio) = Changed from 90% / 10% to 0% / 100% Retention time 17 to 30 min: Liquid A / Liquid B (volume ratio) = 0% / 100% maintained

[0039] Specifically, the high performance liquid chromatography measurement (HPLC measurement) under condition 2 can be carried out in the same manner as under condition 1, except for the following points.

[0040] The measurement sample can be obtained by adjusting the concentration of the scaffold material to 2 mg / mL using a mixture of tetrahydrofuran (THF) and isopropyl alcohol (THF:IPA = 7:3 [volume ratio]) as a solvent. The analysis can be performed, for example, by the following procedure.

[0041] Mobile phase A (liquid A) uses 0.1 wt / vol formic acid methanol, and mobile phase B (liquid B) uses a mixture of THF and IPA (THF:IPA = 7:3 [volume ratio]). At the start of the measurement, mobile phase A is 100% by volume, and from 0 to 2 minutes, mobile phase A is flowed at a constant 100% volume ratio. From 2 to 5 minutes, the proportion of mobile phase B is increased at a constant rate so that the volume ratio of mobile phase A to mobile phase B is 90% / 10% at 5 minutes. From 5 to 17 minutes, the proportion of mobile phase B is increased at a constant rate so that the volume ratio of mobile phase B is 100% at 17 minutes. From 17 to 30 minutes, mobile phase B is flowed at a constant 100% volume ratio. All other points are the same as in Condition 1.

[0042] The first and second inventions of the present application may be implemented either alone or in combination, as described above. Hereinafter, the first and second inventions may be collectively referred to as the present invention.

[0043] The scaffold material according to the present invention has the above-mentioned configuration, and therefore can maintain the stability of cell culture for a long period of time.

[0044] The scaffold material according to the present invention has a relatively high hydrophobicity as determined by HPLC measurement and is appropriately adjusted in hydrophobicity, so that the scaffold material is less likely to dissolve into the liquid medium during cell culture and is less likely to peel off from the container, etc. Therefore, with the scaffold material according to the present invention, the cell proliferation rate is less likely to decrease even when cells are cultured for a long period of time.

[0045] Furthermore, the scaffold material according to the present invention can have good solubility in alcohol solvents such as ethanol. Therefore, for example, a coating liquid in which the scaffold material is dissolved in ethanol can be applied to the surface of a container or the like, and then the ethanol can be evaporated to form a scaffold material layer having a predetermined shape on the surface of the container or the like. Because of its good solubility in alcohol solvents, the concentration of the scaffold material in the coating liquid can be increased, allowing the formation of a thick scaffold material layer.

[0046] Furthermore, the scaffold material according to the present invention does not require the use of natural polymeric materials such as extracellular matrix (ECM) as materials, and therefore is inexpensive, has little variation between lots, and is highly safe.

[0047] In the scaffold material, when the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned condition 1, the retention time of the peak top of the main peak having the largest area among the detected signals is preferably more than 4 minutes, more preferably 5 minutes or more, even more preferably 10 minutes or more, even more preferably 15 minutes or more, and particularly preferably 24 minutes or more. When the retention time of the peak top is equal to or greater than the above-mentioned lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be further increased, and the effects of the present invention can be more effectively exhibited. The retention time of the peak top may be within 30 minutes, but may also exceed 30 minutes. Note that if the main peak is not detected even after 30 minutes, the retention time of the peak top exceeds 30 minutes. Furthermore, in the first invention of the present application, the retention time of the peak top exceeds 4 minutes.

[0048] In the above scaffold material, when the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals is preferably 2 minutes or more, more preferably 5 minutes or more, even more preferably 8 minutes or more, even more preferably 10 minutes or more, even more preferably 15 minutes or more, particularly preferably 18 minutes or more, preferably 30 minutes or less, more preferably 28 minutes or less, even more preferably 27 minutes or less, even more preferably 26 minutes or less, and most preferably 25 minutes or less. When the peak top retention time is above the above-mentioned lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be further increased, and the effects of the present invention can be more effectively exerted. When the peak top retention time is below the above-mentioned upper limit, the solubility in alcohol solvents such as ethanol can be further improved, thereby further improving coatability and processability. In the second invention of the present application, the peak top retention time is 5 minutes or more and 30 minutes or less.

[0049] When a scaffold material is subjected to HPLC measurement under the above-mentioned Condition 1 or Condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals can be adjusted, for example, by the structure, molecular weight, etc. of the peptide-containing (meth)acrylic copolymer. Specifically, as described below, the retention time can be extended by increasing the proportion of hydrophobic (meth)acrylate compounds in the peptide-containing (meth)acrylic copolymer or by appropriately increasing the molecular weight of the peptide-containing (meth)acrylic copolymer.

[0050] The scaffold material will be further described in detail below. In this specification, "(meth)acrylic" means one or both of "acrylic" and "methacrylic", and "(meth)acrylate" means one or both of "acrylate" and "methacrylate".

[0051] (Peptide-containing (meth)acrylic copolymer) The scaffold material contains a peptide-containing (meth)acrylic copolymer. The peptide-containing (meth)acrylic copolymer is a (meth)acrylic copolymer to which a peptide is bound. The peptide-containing (meth)acrylic copolymer has a (meth)acrylic copolymer portion and a peptide portion bound to the (meth)acrylic copolymer portion. Only one type of the peptide-containing (meth)acrylic copolymer may be used, or two or more types may be used in combination.

[0052] <(Meth)acrylic copolymer portion> The (meth)acrylic copolymer portion preferably has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or (A2). This can further increase the hydrophobicity of the peptide-containing (meth)acrylic copolymer. Furthermore, when the scaffold material is subjected to HPLC measurement under the above-mentioned condition 1 or condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals can be increased. The (meth)acrylate compound (A) may contain a (meth)acrylate compound represented by the following formula (A1), may contain a (meth)acrylate compound represented by the following formula (A2), or 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 (meth)acrylate compound (A) contains both a (meth)acrylate compound represented by the following formula (A1) and a (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 (meth)acrylate compound (A) may be used alone or in combination of two or more. 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.

[0053]

[0054] In the above formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0055]

[0056] In the above formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

[0057] R in the above formula (A1) and R in the above formula (A2) may each be an aliphatic hydrocarbon group or an aromatic hydrocarbon group. From the viewpoint of further increasing the solubility of the peptide-containing (meth)acrylic copolymer, it is preferable that R in the above formula (A1) and R in the above formula (A2) each be an aliphatic hydrocarbon group. The aliphatic hydrocarbon group may be linear or may have a branched structure, and may or may not have a double bond. R in the above formula (A1) and R in the above formula (A2) may each be an alkyl group or an alkylene group.

[0058] The number of carbon atoms in R in the above formula (A1) and the number of carbon atoms in R in the above formula (A2) are each 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 carbon numbers are equal to or greater than the above lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be further increased. Furthermore, when the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned condition 1 or condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals can be further increased. When the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned condition 1 or condition 2, the retention time of the peak top of the main peak having the largest area among the detected signals can be further reduced. Furthermore, the solubility of the peptide-containing (meth)acrylic copolymer in alcohol solvents such as ethanol can be further improved, thereby further improving coatability and processability. In particular, when the number of carbon atoms is 12, the effects of the present invention can be even more effectively exhibited, and the coatability and processability can be even more improved.

[0059] In 100 mol% of all structural units in the (meth)acrylic copolymer portion, the content of structural units derived from the (meth)acrylate compound (A) is preferably 25 mol% or more, more preferably 30 mol% or more, even more preferably 40 mol% or more, particularly preferably 50 mol% or more, preferably 98 mol% or less, more preferably 95 mol% or less, even more preferably 90 mol% or less, and particularly preferably 80 mol% or less. In 100 mol% of all structural units in the (meth)acrylic copolymer portion, the content of structural units derived from the (meth)acrylate compound (A) is preferably 25 mol% or more and 98 mol% or less, more preferably 30 mol% or more and 95 mol% or less, even more preferably 40 mol% or more and 90 mol% or less, and particularly preferably 50 mol% or more and 80 mol% or less. When the content is above the lower limit, the hydrophobicity of the peptide-containing (meth)acrylic copolymer can be further increased. Furthermore, when the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned conditions 1 or 2, the retention time of the peak top of the main peak having the largest area among the detected signals can be increased. When the content is equal to or less than the above-mentioned upper limit, the retention time of the peak top of the main peak having the largest area among the detected signals can be decreased when the peptide-containing (meth)acrylic copolymer is subjected to HPLC measurement under the above-mentioned conditions 1 or 2. Furthermore, the solubility of the peptide-containing (meth)acrylic copolymer in alcohol solvents such as ethanol can be further improved, thereby further improving coatability and processability.

[0060] The (meth)acrylic copolymer portion preferably has a structural unit derived from a (meth)acrylate compound (B) having a functional group reactive with an amino group or a carboxyl group. The (meth)acrylate compound (B) may have a functional group reactive with an amino group, a functional group reactive with a carboxyl group, or a functional group reactive with an amino group and a functional group reactive with a carboxyl group. The (meth)acrylate compound (B) may be used alone or in combination of two or more.

[0061] Examples of the functional group capable of reacting with the amino group or carboxyl group include a carboxyl group, a thiol group, an amino group, and a cyano group.

[0062] From the viewpoint of effectively achieving the effects of the present invention, it is preferred that in the peptide-containing (meth)acrylic copolymer, the peptide moiety is bonded to a functional group capable of reacting with the amino group or carboxyl group. More specifically, it is preferred that the carboxyl group or amino group of the amino acid constituting the peptide moiety is bonded to a functional group capable of reacting with the amino group or carboxyl group.

[0063] The functional group capable of reacting with an amino group or a carboxyl group is preferably a carboxyl group or an amino group. The (meth)acrylate compound (B) preferably has a carboxyl group or an amino group.

[0064] Examples of the (meth)acrylate compound (B) include (meth)acrylic acid, 3-butenoic acid, 4-pentenoic acid, 5-hexenoic acid, 6-heptenoic acid, 7-octenoic acid, benzeneacrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxyethyl phthalic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxypropyl phthalic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxyethyl hexahydrophthalic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, and (meth)acryloyloxypropyl hexahydrophthalic acid.

[0065] The (meth)acrylate compound (B) is preferably (meth)acrylic acid, (meth)acryloyloxyethyl succinic acid, (meth)acryloyloxypropyl succinic acid, (meth)acryloyloxyethyl hexahydrosuccinic acid, (meth)acryloyloxypropyl hexahydrosuccinic acid, or butenoic acid, and more preferably (meth)acrylic acid. In this case, the effects of the present invention can be more effectively exhibited.

[0066] The content of structural units derived from the (meth)acrylate compound (B) in 100 mol% of all structural units in the (meth)acrylic copolymer portion is preferably 2 mol% or more, more preferably 5 mol% or more, even more preferably 10 mol% or more, preferably 75 mol% or less, more preferably 70 mol% or less, and even more preferably 60 mol% or less. When the content is above the lower limit, the solubility of the peptide-containing (meth)acrylic copolymer in alcohol solvents can be further increased. When the content is below the upper limit, cell culture stability can be more easily maintained over a long period of time.

[0067] In the total structural units (100 mol%) of the (meth)acrylic copolymer part, the total content of the structural units derived from the (meth)acrylate compound (A) and the structural units derived from the (meth)acrylate compound (B) is preferably 50 mol% or more, more preferably 65 mol% or more, even more preferably 80 mol% or more, even more preferably 90 mol% or more, particularly preferably 95 mol% or more, and most preferably 100 mol%.When the total content is above the lower limit, the effects of the present invention can be more effectively exhibited.In addition, the total content may be 100 mol% or less, or may be 90 mol% or less.

[0068] The (meth)acrylic copolymer portion may contain structural units derived from a (meth)acrylate compound different from both the (meth)acrylate compound (A) and the (meth)acrylate compound (B), provided that the object of the present invention is not violated. Furthermore, the (meth)acrylic copolymer portion may contain structural units derived from a vinyl compound copolymerizable with the (meth)acrylate compound, provided that the object of the present invention is not violated.

[0069] The content of the structural unit derived from the (meth)acrylate compound (A) and the content of the structural unit derived from the (meth)acrylate compound (B) in the (meth)acrylic copolymer portion can be measured, for example, by NMR (nuclear magnetic resonance).

[0070] <Peptide portion> The peptide portion is a structural portion 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 the peptide may be used, or two or more types may be used in combination.

[0071] 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, and preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less. When the number of amino acid residues is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. However, the number of amino acid residues in the peptide portion may be more than 10 or more than 15.

[0072] The peptide portion preferably has a cell-adhesive amino acid sequence. The cell-adhesive amino acid sequence refers to an amino acid sequence whose cell-adhesive activity has been confirmed by phage display, Sepharose bead, or plate coating. The phage display method can be, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995, pp. 1189-1196." The Sepharose bead method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477." The plate coating method can be, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477."

[0073] Examples of the cell adhesive amino acid sequence include the RGD sequence (Arg-Gly-Asp), the YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), the PDSGR sequence (Pro-Asp-Ser-Gly-Arg), the HAV sequence (His-Ala-Val), the ADT sequence (Ala-Asp-Thr), and the 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). Examples of the cell-adhesive amino acid sequence include those described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The peptide portion may have only one or more of the cell-adhesive amino acid sequences.

[0074] The cell adhesive amino acid sequence preferably has at least one of the above-mentioned cell adhesive amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, even more preferably has an RGD sequence, and particularly preferably has at least an RGD sequence represented by the following formula (1): In this case, adhesiveness to cells after seeding can be further increased, and the cell proliferation rate can be further increased.

[0075] Arg-Gly-Asp-X...Formula (1)

[0076] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0077] The peptide portion may be linear or may have a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. From the viewpoint of more effectively exerting the effects of the present invention, the cyclic peptide backbone is preferably composed of four or more amino acids, more preferably five or more amino acids, and more preferably ten or less amino acids.

[0078] In the peptide-containing (meth)acrylic copolymer, the content of the peptide moiety is preferably 0.5 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, preferably 25 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. When the content of the peptide moiety is equal to or greater than the lower limit, the adhesiveness to cells after seeding can be further improved, and the cell proliferation rate can be further increased. When the content of the peptide moiety is equal to or less than the upper limit, the cell culture stability can be further improved and production costs can be reduced. The content (mol%) of the peptide moiety is the amount of the peptide moiety relative to the sum of the amounts of the respective structural units constituting the peptide-containing (meth)acrylic copolymer.

[0079] The content of the peptide moiety can be measured, for example, by NMR (nuclear magnetic resonance).

[0080] <Other Details of the Peptide-Containing (Meth)Acrylic Copolymer> The number-average molecular weight of the peptide-containing (meth)acrylic copolymer is preferably 5,000 or more, more preferably 10,000 or more, even more preferably 50,000 or more, and preferably 5,000,000 or less, more preferably 2,500,000 or less, and even more preferably 1,000,000 or less. When the number-average molecular weight is equal to or greater than the lower limit, the solubility of the peptide-containing (meth)acrylic copolymer in water can be further reduced, and the effects of the present invention can be more effectively exhibited. When the number-average molecular weight is equal to or less than the upper limit, the solubility in alcohol solvents can be further increased.

[0081] The number average molecular weight of the peptide-containing (meth)acrylic copolymer can be measured, for example, by the following method. The peptide-containing (meth)acrylic copolymer is dissolved in tetrahydrofuran (THF) to prepare a 0.2 wt % solution of the peptide-containing (meth)acrylic copolymer. Next, the molecular weight is evaluated using a gel permeation chromatography (GPC) measuring device (APC system, manufactured by Waters) under the following measurement conditions.

[0082] Column: HSPgel HR MB-M 6.0 mm x 150 mm Flow rate: 0.5 mL / min Column temperature: 40°C Injection volume: 10 μL Detector: RI, PDA Standard sample: polystyrene

[0083] The method for producing the peptide-containing (meth)acrylic copolymer is not particularly limited. The peptide-containing (meth)acrylic copolymer can be produced, for example, as follows.

[0084] A monomer mixture containing a (meth)acrylate compound (A) and a (meth)acrylate compound (B) is polymerized to obtain a (meth)acrylate copolymer, and the resulting (meth)acrylate copolymer is reacted with a peptide to obtain a peptide-containing (meth)acrylic copolymer.

[0085] (Other details of the scaffold material for cell culture) The scaffold material is used for culturing cells. The scaffold material is used as a scaffold for the cells when culturing the cells.

[0086] The cells include animal cells from humans, mice, rats, pigs, cows, monkeys, etc. The cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The somatic cells may be cancer cells.

[0087] Examples of the stem cells include somatic stem cells and embryonic stem cells, such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0088] Examples of the mature cells include nerve cells, cardiac muscle cells, retinal cells, and hepatic cells.

[0089] The scaffold material is preferably used for two-dimensional culture (plate culture), three-dimensional culture, or suspension culture of cells, more preferably for two-dimensional culture (plate culture) or three-dimensional culture, and even more preferably for two-dimensional culture.

[0090] The scaffold material is preferably used for serum-free culture. Because the scaffold material contains the peptide-containing (meth)acrylic copolymer, it can enhance cell adhesion even in serum-free culture without feeder cells or adhesive proteins, and in particular, it can enhance the initial cell fixation rate after cell seeding. Furthermore, because the scaffold material contains the peptide-containing (meth)acrylic copolymer, it can exert the effects of the present invention even in serum-free culture.

[0091] The content of the peptide-containing (meth)acrylic copolymer in 100% by weight of the scaffold material 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 peptide-containing (meth)acrylic copolymer is equal to or greater than the lower limit, the effects of the present invention can be more effectively exhibited. However, the content of the peptide-containing (meth)acrylic copolymer in 100% by weight of the scaffold material may be 100% by weight or less, or may be 98% by weight or less.

[0092] The scaffold material may contain components other than the peptide-containing (meth)acrylic copolymer, such as polyolefin resins, polyether resins, polyvinyl alcohol resins, polyesters, epoxy resins, polyamide resins, polyimide resins, polyurethane resins, polycarbonate resins, polysaccharides, cellulose, polypeptides, and synthetic peptides.

[0093] It is preferable that the scaffold material is substantially free of animal-derived raw materials. By not including any animal-derived raw materials, it is possible to provide a scaffold material that is highly safe and has little variation in quality during production. Note that "substantially free of animal-derived raw materials" means that the content of animal-derived raw materials in the scaffold material is 3% by weight or less. In the scaffold material, it is preferable that the content of animal-derived raw materials in the scaffold material is 1% by weight or less, and most preferably 0% by weight. In other words, it is most preferable that the scaffold material is completely free of animal-derived raw materials.

[0094] The shape of the scaffold material is not particularly limited, and may be in the form of particles, fibers, a porous body, or a membrane.

[0095] The scaffold material is preferably a resin film. The resin film is preferably a resin film formed from the scaffold material. The resin film is a membranous scaffold material.

[0096] The thickness of the resin film is not particularly limited. The average thickness of the resin film may be 10 nm or more, 50 nm or more, or 500 nm or more, and may be 1000 μm or less, or 500 μm or less.

[0097] (Cell culture vessel) The cell culture vessel preferably includes the above-mentioned resin film in at least a part of the cell culture region. The cell culture vessel preferably includes a vessel body and the above-mentioned resin film, and the resin film is preferably disposed on the surface of the vessel body. The resin film is preferably a membranous scaffold material, and is preferably a scaffold material layer.

[0098] FIG. 1 is a cross-sectional view that schematically shows a cell culture vessel according to one embodiment of the present invention.

[0099] The cell culture vessel 1 includes a vessel body 2 and a resin film 3. The resin film 3 is disposed on a surface 2a of the vessel body 2. The resin film 3 is disposed on a bottom surface of the vessel body 2. By adding a liquid medium to the cell culture vessel 1 and seeding cells such as cell clumps on the surface of the resin film 3, cells can be cultured on a plate.

[0100] The container body may include a first container body and a second container body such as a cover glass on the bottom surface of the first container body. The first container body and the second container body may be separable. In this case, a resin film 3 may be disposed on the surface of the second container body.

[0101] As the container body, a conventionally known container body (container) can be used. The shape and size of the container body are not particularly limited. Examples of the container body that can be used include 2- to 384-well plates, single-layer flasks, multi-layer flasks, multi-sided flasks, dishes, roller bottles, bags, insert cups, and microchannel chips.

[0102] The material of the container body is not particularly limited, but examples thereof include synthetic resins, metals, glass, etc. Examples of the synthetic resins include polystyrene, polyethylene, polypropylene, polyethersulfone, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimides, polyamides, polyamideimides, (meth)acrylic resins, epoxy resins, and silicones.

[0103] (Microcarrier for cell culture) Microcarriers for cell culture (hereinafter sometimes abbreviated as "microcarriers") comprise base particles and a coating layer that covers the outer surface of the base particles, and the coating layer is preferably formed from the scaffold material. The coating layer is preferably a scaffold material layer. As the base particles, conventionally known base particles used in microcarriers can be used. Examples of the base particles include resin particles.

[0104] FIG. 2 is a cross-sectional view schematically showing a microcarrier for cell culture according to one embodiment of the present invention.

[0105] The cell culture microcarrier 5 shown in Figure 2 comprises a base particle 6 and a coating layer 7 that coats the outer surface of the base particle 6. The coating layer 7 is disposed on the surface of the base particle 6 and is in contact with the surface of the base particle 6. The coating layer 7 coats the entire outer surface of the base particle 6.

[0106] (Cell culture method) Cells can be cultured using the scaffold material, the resin film, and the microcarriers. The cell culture method is a cell culture method using the scaffold material. The cell culture method is preferably a cell culture method using the resin film, and is preferably a cell culture method using the microcarriers. Examples of the cells include the cells described above.

[0107] The cell culture method preferably comprises a step of seeding cells on the scaffold material. The cell culture method preferably comprises a step of seeding cells on the resin film. The cell culture method preferably comprises a step of seeding cells on the microcarriers. The cells may be cell clumps. The cell clumps can be obtained by adding a cell release agent to a confluent culture vessel and homogenizing the cells by pipetting. The cell release agent is not particularly limited, but ethylenediamine / phosphate buffer solution is preferred. The size of the cell clumps is preferably 50 μm to 200 μm.

[0108] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0109] The content of the structural unit and the content of the peptide moiety in the obtained peptide-containing (meth)acrylic copolymer were measured by dissolving the copolymer in DMSO-d6 (dimethyl sulfoxide) and then using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0110] Example 1 Preparation of peptide-containing (meth)acrylic copolymer: 4.5 parts by weight of butyl acrylate and 7.0 parts by weight of acrylic acid were dissolved in 27 parts by weight of tetrahydrofuran to obtain an acrylic monomer solution. 0.0575 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained acrylic monomer solution, and the obtained solution was applied to a PET film. The coated material was exposed to light of 365 nm wavelength at an integrated light intensity of 2000 mJ / cm using a UV conveyor device ("ECS301G1" manufactured by iGraphics Co., Ltd.) at 25°C. ２ The (meth)acrylic copolymer solution was obtained by irradiating the (meth)acrylic copolymer solution with 1000 nm of UV radiation at 80° C. for 3 hours, and the (meth)acrylic copolymer solution was obtained by vacuum drying the (meth)acrylic copolymer solution at 80° C. for 3 hours.

[0111] A cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (five amino acid residues, Arg and Lys bonded to form a cyclic skeleton, Phe is D-configuration, and is referred to as c-RGDfK in the table) was prepared as the peptide. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared as the condensing agent. 50 parts by weight of a (meth)acrylic copolymer and 10 parts by weight of a peptide were dissolved in 1,000 parts by weight of DMF (N,N-dimethylformamide) to prepare a first solution. Furthermore, 1 part by weight of the condensing agent was mixed with 1,000 parts by weight of DMF to prepare a second solution. The first and second solutions were mixed to prepare a solution containing the (meth)acrylic copolymer, peptide, and condensing agent. The resulting solution was reacted at 40°C for 2 hours to cause dehydration condensation between the carboxyl group in the structural unit derived from acrylic acid of the (meth)acrylic copolymer and the amino group of Lys of the peptide, thereby obtaining a solution containing a peptide-containing (meth)acrylic copolymer.

[0112] Preparation of coating solution: The resulting solution containing the peptide-containing (meth)acrylic copolymer was diluted 100-fold with DMF and washed by dripping it into a column packed with ion exchange resin (manufactured by Organo Corporation) at a rate of 0.3 mL / min. The washed solution was vacuum dried at 60°C for 3 hours, and the resulting dry solid was dissolved in ethanol to obtain a coating solution containing the peptide-containing (meth)acrylic copolymer and ethanol. The content of the peptide-containing (meth)acrylic copolymer in the coating solution was 0.1 wt%.

[0113] Fabrication of cell culture vessel: 20 μL of the obtained coating liquid was applied to each well of a 6-well plate by cast coating, and then the ethanol was removed by vacuum drying at 60° C. for 3 hours. In this way, a cell culture vessel was obtained in which a resin film (membrane-like scaffold material) formed from the scaffold material was placed on the bottom surface of each well.

[0114] (Examples 2 to 10 and Comparative Examples 1 to 6) Peptide-containing (meth)acrylic copolymers having the structures shown in Tables 1 to 3 were obtained in the same manner as in Example 1, except that the type and amount of the (meth)acrylate compound were changed. Cell culture vessels were also obtained in the same manner as in Example 1, except that the obtained peptide-containing (meth)acrylic copolymers were used. In Comparative Examples 1 and 3, light acrylate (MTG-A, manufactured by Kyoeisha Chemical Co., Ltd., methoxy-triethylene glycol acrylate) was used in the formulation, and in Comparative Example 7, Synthemax vitronectin substrate (manufactured by Corning Incorporated) was used instead of the peptide-containing (meth)acrylic copolymer.

[0115] (Evaluation) (1) Number-Average Molecular Weight of Peptide-Containing (Meth)acrylic Copolymer The number-average molecular weight of the resulting peptide-containing (meth)acrylic copolymer was measured by the method described above.

[0116] (2) Peak Top Retention Time (Condition 1) HPLC measurement under condition 1 was carried out as follows, and the peak top retention time of the main peak having the largest area among the detected signals was determined.

[0117] The measurement sample was obtained by adjusting the peptide-containing (meth)acrylic copolymer (a resin film made of a scaffold material) to a concentration of 2 mg / mL using a mixture of 0.1 wt% aqueous formic acid and isopropyl alcohol (0.1 wt% aqueous formic acid / isopropyl alcohol = 7 / 3 [volume ratio]) as a solvent.

[0118] The measuring equipment used was an HPLC device (Shimadzu Corporation, High-Pressure Gradient HPLC System Prominence), an HPLC column (Waters Corporation, XBridge (registered trademark) C18 [internal diameter 3.0 mm × length 150 mm, packing particle size 3.5 μm]), and an evaporative light scattering detector (Shimadzu Corporation, ELSD_LTII). The analysis was performed according to the following procedure.

[0119] A 0.1 wt % formic acid aqueous solution was used as mobile phase A (liquid A), and isopropyl alcohol was used as mobile phase B (liquid B). At the start of the measurement, the inside of the HPLC apparatus was filled with a mixed solvent of mobile phase A / mobile phase B in a volume ratio of 7 / 3. In this state, the measurement sample (injection volume: 20 μL) was injected. Then, immediately after sample injection, the proportion of mobile phase B in the mobile phase was increased at a constant rate over 15 minutes so that the proportion of mobile phase B in the mobile phase reached 100% by volume. After 15 minutes (at this point, the mobile phase was completely replaced with mobile phase B), mobile phase B was allowed to flow for 15 minutes. The column temperature was 40°C, and the total flow rate was 0.3 mL / min.

[0120] Nitrogen gas was used as the nebulizer gas for the evaporative light scattering detector. The gas supply pressure was 350 kPa, and the drift tube temperature was 40 ° C. The baseline was determined by analyzing a blank test solution prepared in the same manner as the preparation of the analytical sample, except that the scaffold material was dissolved.

[0121] (3) Peak Top Retention Time (Condition 2) HPLC measurement under condition 2 was carried out in the same manner as under condition 1, except for the following points, and the retention time of the peak top of the main peak having the largest area among the detected signals was determined.

[0122] The measurement sample was prepared by adjusting the peptide-containing (meth)acrylic copolymer (a resin film made of a scaffold material) to a concentration of 2 mg / mL using a mixture of THF and IPA (THF:IPA = 7:3 (volume ratio)) as a solvent. The analysis was carried out as follows.

[0123] Mobile phase A (liquid A) was 0.1 wt / vol formic acid methanol, and mobile phase B (liquid B) was a mixture of THF and isopropyl alcohol (THF:IPA = 7:3 [volume ratio]). At the start of the measurement, mobile phase A was 100% by volume, and from 0 to 2 minutes, mobile phase A was flowed at a constant volume ratio of 100%. From 2 to 5 minutes, the proportion of mobile phase B was increased at a constant rate so that the volume ratio of mobile phase A / mobile phase B was 90% / 10% at the 5-minute mark. From 5 to 17 minutes, the proportion of mobile phase B was increased at a constant rate so that the volume ratio of mobile phase B was 100% at the 17-minute mark. From 17 to 30 minutes, mobile phase B was flowed at a constant volume ratio of 100%.

[0124] (4) Cell Culture Stability (Long-Term Culture Stability) The following liquid media were prepared.

[0125] R-STEM (Rohto Pharmaceutical Co., Ltd.)

[0126] 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the vessel was allowed to stand in an incubator at 37° C. for 1 hour, after which the phosphate buffered saline was removed from the cell culture vessel.

[0127] 5 x 10 in 1.5 mL of liquid medium ４ A cell suspension containing 100 cells (Lonza, human adipose-derived mesenchymal stem cells, model number: PT-5006) was prepared. This cell suspension was seeded into each well of a 6-well plate. The 6-well plate was then shaken left and right five times and incubated at 37°C and CO ２ The mixture was cultured in an incubator containing 5% of the lysate.

[0128] The number of cells 24 hours, 48 ​​hours, and 72 hours after seeding was counted using a NucleoCounter NC-3000 (MS Techno Systems). T1 was the cell doubling time between 24 and 48 hours after seeding, and T2 was the cell doubling time between 48 and 72 hours after seeding. T1 and T2 were calculated using the following formulas.

[0129] T1=24log2 / (log(N(48) / N(24)) T2=24log2 / (log(N(72) / N(48))

[0130] T1: Cell doubling time 24 to 48 hours after seeding T2: Cell doubling time 48 to 72 hours after seeding N(24): Number of cells 24 hours after seeding N(48): Number of cells 48 hours after seeding N(72): Number of cells 72 hours after seeding

[0131] The ratio of T1 to T2 (T1 / T2) was calculated from the doubling time (T1) and the doubling time (T2). The cell culture stability was evaluated according to the following criteria: a larger ratio (T1 / T2) means that the cell proliferation speed has not decreased.

[0132] <Criteria for determining cell culture stability (long-term culture stability)> ○○: Ratio (T1 / T2) is 0.9 or more ○: Ratio (T1 / T2) is 0.7 or more but less than 0.9 ×: Ratio (T1 / T2) is less than 0.7

[0133] 3 shows phase-contrast micrographs taken 24 hours, 48 ​​hours, and 72 hours after cell seeding when cell culture was performed using the scaffold materials for cell culture obtained in Examples 1 and 8 and Comparative Example 2. In Example 1, the ratio (T1 / T2) was 0.85, in Example 8, the ratio (T1 / T2) was 0.99, and in Comparative Example 2, the ratio (T1 / T2) was 0.5.

[0134] (5) Solubility in Ethanol The solubility of the obtained scaffold material in ethanol was measured by the following method. The scaffold material was mixed with ethanol and stirred at 60 ° C. After 30 minutes, the presence or absence of residual scaffold material and the transparency of the solution were visually confirmed. The maximum solution concentration at which there was no residual scaffold material and the solution was transparent was defined as the solubility. The solubility in ethanol was evaluated according to the following criteria.

[0135] <Criteria for determining solubility in ethanol> ○○: Solubility is 1% by weight or more ○: Solubility is 0.1% by weight or more but less than 1% by weight ×: Solubility is less than 0.1% by weight

[0136] Details and results are shown in Tables 1 to 3 below.

[0137]

[0138]

[0139]

[0140] DESCRIPTION OF SYMBOLS 1... Cell culture vessel 2... Vessel body 2a... Surface 3... Resin film 5... Cell culture microcarrier 6... Base particle 7... Coating layer

Claims

1. A scaffold material for cell culture, comprising: The present invention comprises a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bonded to the (meth)acrylic copolymer portion, A cell culture scaffold material in which, when the peptide-containing (meth)acrylic copolymer is measured by high-performance liquid chromatography under the following condition 1, the peak top of the main peak with the largest area among the signals is not detected within a retention time of 4 minutes. [Condition 1] Column: C18 (inner diameter 3.0 mm x length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3mL / min Detector: Evaporative light scattering detector Mobile phase A (Solution A): 0.1% by weight formic acid aqueous solution Mobile phase B (Solution B): Isopropyl alcohol Holding time 0 minutes: Solution A / Solution B (volume ratio) = 70% / 30% Holding time 0 to 15 minutes: Solution A / Solution B (volume ratio) changes from 70% / 30% to 0% / 100%. Holding time 15-30 minutes: Maintain a volume ratio of 0% / 100% for Solution A / Solution B.

2. The cell culture scaffold material according to claim 1, wherein when the peptide-containing (meth)acrylic copolymer is measured by high-performance liquid chromatography under condition 1, the retention time of the peak top of the main peak with the largest area among the detected signals is 5 minutes or more and 30 minutes or less.

3. A scaffold material for cell culture, comprising: The present invention comprises a peptide-containing (meth)acrylic copolymer having a (meth)acrylic copolymer portion and a peptide portion bonded to the (meth)acrylic copolymer portion, A cell culture scaffold material wherein, when the peptide-containing (meth)acrylic copolymer is measured by high-performance liquid chromatography under the conditions described in item 2 below, the retention time of the peak top of the main peak with the largest area among the detected signals is between 5 minutes and 30 minutes. [Condition 2] Column: C18 (inner diameter 3.0 mm x length 150 mm, packing particle size 3.5 μm) Column temperature: 40°C Flow rate: 0.3mL / min Detector: Evaporative light scattering detector Mobile phase A (Solution A): 0.1 wt / vol methanol formate Mobile phase B (Solution B): THF / Isopropyl alcohol = 7 / 3 (volume ratio) Holding time 0-2 minutes: Maintain a volume ratio of 100% for Solution A and 0% for Solution B. Holding time 2 to 5 minutes: Solution A / Solution B (volume ratio) changes from 100% / 0% to 90% / 10%. Holding time 5 to 17 minutes: Solution A / Solution B (volume ratio) changes from 90% / 10% to 0% / 100%. Holding time 17-30 minutes: Maintain a volume ratio of 0% / 100% for Solution A / Solution B.

4. The scaffold material for cell culture according to any one of claims 1 to 3, wherein the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (A) represented by the following formula (A1) or the following formula (A2): 【Chemistry 1】 In the formula (A1), R represents a hydrocarbon group having 2 to 18 carbon atoms. 【Chemistry 2】 In the formula (A2), R represents a hydrocarbon group having 2 to 18 carbon atoms.

5. In formula (A1), the number of carbon atoms in R is 6 or more, The cell culture scaffold material according to claim 4, wherein the number of carbon atoms in R in formula (A2) is 6 or more.

6. the (meth)acrylic copolymer portion has a structural unit derived from a (meth)acrylate compound (B) having a functional group capable of reacting with an amino group or a carboxyl group, The scaffold material for cell culture according to any one of claims 1 to 3, wherein in the peptide-containing (meth)acrylic copolymer, the peptide moiety is bonded to a functional group capable of reacting with the amino group or the carboxyl group.

7. 5. The scaffold material for cell culture according to claim 4, wherein the content of structural units derived from the (meth)acrylate compound (A) is 25 mol% or more and 98 mol% or less out of 100 mol% of all structural units in the (meth)acrylic copolymer portion.

8. The (meth)acrylic copolymer portion has a structural unit derived from the (meth)acrylate compound (A) and a structural unit derived from a (meth)acrylate compound (B) having a functional group capable of reacting with an amino group or a carboxyl group, In the peptide-containing (meth)acrylic copolymer, the peptide portion is bonded to a functional group capable of reacting with the amino group or the carboxyl group, the content of structural units derived from the (meth)acrylate compound (A) is 25 mol % or more and 98 mol % or less, based on 100 mol % of all structural units in the (meth)acrylic copolymer portion; the content of structural units derived from the (meth)acrylate compound (B) is 2 mol % or more and 75 mol % or less, based on 100 mol % of all structural units in the (meth)acrylic copolymer portion; 5. The scaffold material for cell culture according to claim 4, wherein the total content of structural units derived from the (meth)acrylate compound (A) and structural units derived from the (meth)acrylate compound (B) is 95 mol % or more, based on 100 mol % of all structural units in the (meth)acrylic copolymer portion.

9. The cell culture scaffold material according to any one of claims 1 to 3, wherein the number average molecular weight of the peptide-containing (meth)acrylic copolymer is 5,000 or more.

10. The cell culture scaffold material according to any one of claims 1 to 3, wherein the peptide-containing (meth)acrylic copolymer has a peptide content of 0.5 mol% or more and 25 mol% or less.

11. The cell culture scaffold material according to any one of claims 1 to 3, wherein the peptide portion has an RGD sequence.

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