Scaffolding material for cell culture, resin film, container for cell culture, and method for culturing cells

Abstract

To provide a scaffold material for cell culture that can increase adhesiveness of cells.SOLUTION: A scaffold material for cell culture according to the present invention contains peptide-containing acrylic resin, and the peptide-containing acrylic resin has a first structural part which does not have a peptide part in a side chain, and a second structural part which has a peptide part in a side chain. A solubility parameter calculated by a formula of Okitsu of the first structural part is 9.7 (cal / cm3)1 / 2 or more and 10.7 (cal / cm3)1 / 2 or less.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a scaffold material for cell culture. The present invention also relates to a resin membrane using the scaffold material for cell culture. The present invention further relates to a cell culture vessel and a cell culture method using the resin membrane. [Background technology]

[0002] Animal cells such as those from humans, mice, rats, pigs, cattle, and monkeys are used in research and development in the fields of academics, drug discovery, regenerative medicine, etc. As scaffold materials for culturing animal cells, adhesive proteins such as laminin and vitronectin, and natural polymer materials such as Matrigel derived from mouse sarcoma are used.

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

[0004] For example, 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 to each other. In Patent Document 1, a hydrophilic acrylic polymer in which a hydrophilic acrylic monomer is polymerized is used as the acrylic polymer. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2012 / 158235A2 Summary of the Invention [Problem to be solved by the invention]

[0006] A scaffold material for cell culture using an acrylic resin bound to a peptide is known, as described in Patent Document 1. However, with the scaffold material described in Patent Document 1, it is difficult to sufficiently increase the adhesiveness of cells.

[0007] An object of the present invention is to provide a scaffold material for cell culture capable of enhancing cell adhesion. Another object of the present invention is to provide a resin film using the scaffold material for cell culture. A further object of the present invention is to provide a cell culture vessel and a cell culture method using the resin film. [Means for solving the problem]

[0008] According to a broad aspect of the present invention, there is provided a peptide-containing acrylic resin, the peptide-containing acrylic resin having a first structural part having no peptide moiety in a side chain and a second structural part having a peptide moiety in a side chain, the first structural part having a solubility parameter of 9.7 (cal / cm) calculated by Okitsu's formula. 3 ) 1 / 2 Above, 10.7(cal / cm 3 ) 1 / 2 The following cell culture scaffold material is provided:

[0009] In a specific aspect of the scaffold material for cell culture according to the present invention, the first structural portion has a poly(meth)acrylic acid ester skeleton.

[0010] In a specific aspect of the scaffold material for cell culture according to the present invention, the number of amino acid residues in the peptide portion in the second structural portion is 10 or less.

[0011] According to a broad aspect of the present invention, a resin film formed from the above-mentioned scaffold material for cell culture is Provided.

[0012] In a specific aspect of the resin film according to the present invention, after immersion in ion-exchanged water at 37°C for 24 hours, the compressive modulus at a frequency of 1 Hz measured in the ion-exchanged water using a nanoindenter device in accordance with ISO14577-1 is 1 GPa or more.

[0013] In a specific aspect of the resin film according to the present invention, the water swelling ratio is 70% or less.

[0014] In a specific aspect of the resin film according to the present invention, the resin film has an island-in-a-sea structure, and the islands in the island-in-a-sea structure contain the peptide portion.

[0015] According to a broad aspect of the present invention, there is provided a cell culture vessel comprising the above-mentioned resin membrane in at least a portion of a cell culture region.

[0016] According to a broad aspect of the present invention, there is provided a method for culturing cells, comprising the step of seeding cells on the above-described resin film. Effect of the Invention

[0017] The scaffold material for cell culture according to the present invention comprises a peptide-containing acrylic resin, and the peptide-containing acrylic resin has a first structural part that does not have a peptide moiety in a side chain and a second structural part that has a peptide moiety in a side chain. In the scaffold material for cell culture according to the present invention, the solubility parameter of the first structural part calculated by Okitsu's formula is 9.7 (cal / cm 3 ) 1 / 2 Above, 10.7(cal / cm 3 ) 1 / 2 The scaffold material for cell culture according to the present invention has the above-mentioned configuration, and therefore can enhance the adhesiveness of cells. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a cross-sectional view that illustrates a cell culture vessel according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail below.

[0020] The scaffold material for cell culture according to the present invention (hereinafter sometimes abbreviated as "scaffold material") contains a peptide-containing acrylic resin, and the peptide-containing acrylic resin has a first structural part that does not have a peptide moiety in its side chain, and a second structural part that has a peptide moiety in its side chain. In the scaffold material for cell culture according to the present invention, the solubility parameter of the first structural part calculated by Okitsu's formula is 9.7 (cal / cm 3 ) 1 / 2 Above, 10.7(cal / cm 3 ) 1 / 2 The following is the result.

[0021] Since the scaffold material according to the present invention has the above-mentioned configuration, it is possible to enhance the adhesiveness of cells. The scaffold material according to the present invention can maintain high adhesiveness of cells for a long period of time.

[0022] It is difficult to sufficiently increase cell adhesion in conventional scaffold materials using peptide-bound acrylic resins (peptide-containing acrylic resins). The present inventors have found that the reason why cell adhesion cannot be sufficiently increased in conventional scaffold materials using peptide-containing acrylic resins is because a highly hydrophilic acrylic resin is used. The present inventors have found that in peptide-bound acrylic resins, the solubility parameter (SP value) of a specific structural portion is set within a specific range, and the balance between the hydrophilicity and hydrophobicity of the structural portion is improved, thereby increasing cell adhesion.

[0023] In addition, since conventional scaffold materials use highly hydrophilic acrylic resins, the scaffold material swells excessively during cell culture, causing the scaffold material to peel off from the container, etc., and the cell adhesion varies from lot to lot, making it difficult to use for mass cell culture. In contrast, the scaffold material of the present invention is less likely to peel off from the container, etc. during cell culture, and the performance variation from lot to lot can be suppressed, so it can also be used for mass cell culture.

[0024] (Peptide-containing acrylic resin) The scaffold material includes a peptide-containing acrylic resin. The peptide-containing acrylic resin is an acrylic resin to which a peptide is bound. The peptide-containing acrylic resin has a main chain and a side chain. The peptide-containing acrylic resin has a portion (first structural portion) that does not have a peptide moiety in the side chain, and a portion (second structural portion) that has a peptide moiety in the side chain. Only one type of the peptide-containing acrylic resin may be used, or two or more types may be used in combination.

[0025] The first structural portion is a structural portion of the peptide-containing acrylic resin other than the second structural portion. For example, when the peptide-containing acrylic resin is composed of M structural units (A) having no peptide portion and N structural units (B) having a peptide portion, the first structural portion is a portion composed of M structural units (A), and the second structural portion is a portion composed of N structural units (B). The structural unit (A) may be only one type of structural unit, or may be two or more types of structural units. The structural unit (B) may be only one type of structural unit, or may be two or more types of structural units. In addition, the structural units (A) and the structural units (B) may be present alternately, randomly, or in blocks in the peptide-containing acrylic resin, or some structural units may be grafted.

[0026] In this specification, "(meth)acrylic" means either or both of "acrylic" and "methacrylic", and "(meth)acrylate" means either or both of "acrylate" and "methacrylate".

[0027] <First structural part> The first structural portion is a structural portion that does not have a peptide portion in the side chain in the peptide-containing acrylic resin. The first structural portion is preferably a portion that is composed of a structural unit that does not have a peptide portion in the peptide-containing acrylic resin. The first structural portion preferably has a structural unit that does not have a peptide portion as a repeating structural unit.

[0028] The first structural portion preferably has a structural unit derived from a (meth)acrylic acid ester. The first structural portion may have only a structural unit derived from a (meth)acrylic acid ester. The first structural portion preferably has a poly(meth)acrylic acid ester skeleton.

[0029] The first structural portion may or may not have a structural unit derived from a monomer other than a (meth)acrylic acid ester.

[0030] The types of the (meth)acrylic acid ester and the monomer other than the (meth)acrylic acid ester are not particularly limited. In the present invention, the types of the (meth)acrylic acid ester and the monomer other than the (meth)acrylic acid ester are appropriately selected so that the solubility parameter (SP value) of the first structure portion satisfies a specific range.

[0031] Examples of the (meth)acrylic acid ester include (meth)acrylic acid alkyl ester, (meth)acrylic acid cyclic alkyl ester, (meth)acrylic acid aryl ester, (meth)acrylic acid polyethylene glycol, (meth)acrylic acid phosphorylcholine, etc. The (meth)acrylic acid ester may be used alone or in combination of two or more kinds.

[0032] Examples of the (meth)acrylic acid alkyl ester 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.

[0033] The (meth)acrylic acid alkyl ester may be substituted with a substituent such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples of such (meth)acrylic acid alkyl ester include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0034] Examples of the (meth)acrylic acid cyclic alkyl ester include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0035] Examples of the (meth)acrylic acid aryl ester include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0036] Examples of the polyethylene glycol (meth)acrylates 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.

[0037] The (meth)acrylate phosphorylcholine includes 2-(meth)acryloyloxyethyl phosphorylcholine and the like.

[0038] Examples of the monomer other than the (meth)acrylic acid ester include (meth)acrylamides and vinyl compounds.

[0039] The monomers other than the (meth)acrylic acid esters may be used alone or in combination of two or more kinds.

[0040] Examples of the (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, and the like. amide, N-(2-hydroxyethyl)(meth)acrylamide, N-methylol(meth)acrylamide, and 6-(meth)acrylamide hexanoic acid.

[0041] Examples of the vinyl compound include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, and vinylamine.

[0042] From the viewpoint of easily adjusting the solubility parameter (SP value), the first structural portion preferably has structural units derived from two or more kinds of (meth)acrylic acid esters.

[0043] The solubility parameter (SP value) of the first structural portion calculated by the Okitsu formula is 9.7 (cal / cm 3 ) 1 / 2 Above, 10.7(cal / cm 3 ) 1 / 2 The solubility parameter (SP value) is 9.7 (cal / cm 3 ) 1 / 2 Less than or equal to 10.7 (cal / cm 3 ) 1 / 2 If the solubility parameter (SP value) exceeds 10.7 (cal / cm), it is difficult to increase the adhesiveness of cells, and it is particularly difficult to increase the adhesiveness of cells over a long period of time. 3 ) 1 / 2 If the temperature exceeds this range, the scaffold material may peel off from the container during cell culture.

[0044] The solubility parameter (SP value) of the first structural portion calculated by the Okitsu formula is preferably 9.8 (cal / cm 3 ) 1 / 2 More preferably, 10.0 (cal / cm 3 ) 1 / 2 More preferably, 10.2 (cal / cm 3 ) 1 / 2 The solubility parameter (SP value) of the first structural portion calculated by the Okitsu formula is preferably 10.5 (cal / cm 3 ) 1 / 2 Less than or equal to 10.3 (cal / cm 3 ) 1 / 2 More preferably, 10.2 (cal / cm 3 ) 1 / 2 When the solubility parameter (SP value) is equal to or greater than the lower limit and equal to or less than the upper limit, the adhesiveness of cells can be further improved. When the solubility parameter (SP value) is equal to or less than the upper limit, the scaffold material can be effectively prevented from peeling off from a container or the like during cell culture.

[0045] More specifically, the solubility parameter (SP value) is calculated using Okitsu's ΔF constant (Okitsu Toshinao, Adhesion, Vol. 40, No. 8, p. 342 (1996)).

[0046] In 100 mol% of all structural units contained in the first structural portion, the average ratio of the structural units derived from the (meth)acrylic acid ester is preferably 20 mol% or more, more preferably 30 mol% or more, even more preferably 40 mol% or more, even more preferably 50 mol% or more, even more preferably 60 mol% or more, and particularly preferably 70 mol% or more. When the average ratio of the structural units derived from the (meth)acrylic acid ester is equal to or more than the lower limit, the solubility parameter (SP value) is easily adjusted, and the effects of the present invention can be more effectively exhibited. In 100 mol% of all structural units contained in the first structural portion, the average ratio of the structural units derived from the (meth)acrylic acid ester may be 80 mol% or more, 90 mol% or more, 95 mol% or more, or even 100 mol%. In 100 mol% of all structural units contained in the first structural portion, the average ratio of the structural units derived from the (meth)acrylic acid ester may be 100 mol% or less, or 90 mol% or less.

[0047] In 100% by weight of the peptide-containing acrylic resin, the content of the first structural portion is preferably 20% by weight or more, more preferably 30% by weight or more, even more preferably 40% by weight or more, preferably 95% by weight or less, more preferably 90% by weight or less, and even more preferably 85% by weight or less. When the content of the first structural portion is equal to or more than the lower limit and equal to or less than the upper limit, the effects of the present invention can be more effectively exhibited.

[0048] In 100% by weight of the peptide-containing acrylic resin, the content of the structural unit derived from the (meth)acrylic acid ester is preferably 20% by weight or more, more preferably 30% by weight or more, even more preferably 40% by weight or more, preferably 95% by weight or less, more preferably 90% by weight or less, and even more preferably 85% by weight or less. When the content of the structural unit derived from the (meth)acrylic acid ester is equal to or more than the lower limit and equal to or less than the upper limit, the effects of the present invention can be more effectively exhibited.

[0049] <Second structural part> The second structural portion is a structural portion having a peptide portion in a side chain in the peptide-containing acrylic resin. The second structural portion is a portion of the peptide-containing acrylic resin that is composed of a structural unit having a peptide portion. The second structural portion preferably has a structural unit having a peptide portion as a repeating structural unit.

[0050] The second structural portion preferably has a structural unit having a backbone derived from a peptide and a backbone derived from a compound having a functional group capable of binding to the peptide. The structural unit having a peptide portion preferably has a backbone derived from a peptide and a backbone derived from a compound having a functional group capable of binding to the peptide.

[0051] In this specification, the above-mentioned "compound having a functional group capable of binding to a peptide" may be referred to as "compound X."

[0052] Therefore, the second structural portion preferably has a structural unit having a skeleton derived from a peptide and a skeleton derived from compound X. The structural unit having the peptide portion preferably has a skeleton derived from a peptide and a skeleton derived from compound X. In the structural unit having a skeleton derived from a peptide and a skeleton derived from compound X, the peptide and compound X are bonded. Only one type of the peptide and the compound X may be used, or two or more types may be used in combination.

[0053] The functional group capable of binding to the peptide is preferably a functional group capable of condensing with a carboxyl group or an amino group of the peptide.

[0054] Examples of the functional group capable of binding to the peptide include a carboxyl group, a thiol group, an amino group, a hydroxyl group, and a cyano group.

[0055] The compound X preferably has a functional group capable of bonding to a (meth)acrylic acid ester.

[0056] Examples of the functional group capable of bonding to the (meth)acrylic acid ester include a vinyl group, a (meth)acryloyl group, and an allyl group.

[0057] The compound X preferably has a carboxyl group or an amino group as a functional group capable of bonding to the peptide, and more preferably has a (meth)acryloyl group as a functional group capable of bonding to the (meth)acrylic acid ester.

[0058] The compound X is preferably a compound having a carboxyl group or an amino group and a (meth)acryloyl group.

[0059] Examples of the compound X include (meth)acrylic acid, itaconic acid, acrylamide, etc. The compound X may be used alone or in combination of two or more kinds.

[0060] The compound X is preferably (meth)acrylic acid or itaconic acid, and more preferably (meth)acrylic acid.

[0061] The number of amino acid residues in the peptide portion in the second structural portion is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, 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 more than the lower limit and equal to or less than the upper limit, the adhesiveness to cells after seeding can be further increased, 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.

[0062] 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 the phage display method, the Sepharose bead method, or the plate coating method. As the phage display method, for example, the method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 pp. 1189-1196" can be used. As the Sepharose bead method, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477" can be used. As the plate coating method, for example, the method described in "Protein, Nucleic Acid, Enzyme, Vol. 45, No. 15 (2000) 2477" can be used.

[0063] Examples of the cell adhesive amino acid sequences include the 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). Further, examples of the cell adhesive amino acid sequence include the sequences 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 type of cell adhesive amino acid sequence, or two or more types.

[0064] The cell adhesive amino acid sequence preferably has at least any 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, and even more preferably has at least an RGD sequence represented by the following formula (1). In this case, the adhesiveness to cells after seeding can be further increased, and the cell proliferation rate can be further increased.

[0065] Arg-Gly-Asp-X...Formula (1)

[0066] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0067] The peptide portion may be linear or 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 4 or more amino acids, more preferably composed of 5 or more amino acids, and more preferably composed of 10 or less amino acids.

[0068] In the peptide-containing acrylic resin, the content of the peptide moiety is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, and particularly preferably 10 mol% or more. In the peptide-containing acrylic resin, the content of the peptide moiety is 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 moiety is equal to or more than the lower limit, the phase separation structure can be formed more easily. When the content of the peptide moiety is equal to or more than the lower limit, the adhesiveness to the cells after seeding can be further improved, and the cell proliferation rate can be further improved. In addition, when the content of the peptide moiety is equal to or less than the upper limit, the production cost can be reduced. The content of the peptide moiety (mol%) is the amount of substance of the peptide moiety relative to the sum of the amounts of substances of each structural unit constituting the synthetic resin having the peptide moiety.

[0069] The content of the above peptide portion can be measured by FT-IR or LC-MS.

[0070] In 100 mol % of the peptide-containing acrylic resin, the content of the second structural portion is preferably 0.1 mol % or more, more preferably 0.5 mol % or more, even more preferably 1 mol % or more, preferably 60 mol % or less, more preferably 50 mol % or less, and even more preferably 35 mol % or less. When the content of the second structural portion is equal to or more than the lower limit and equal to or less than the upper limit, the effects of the present invention can be more effectively exhibited.

[0071] <Other details of peptide-containing acrylic resin> The weight-average molecular weight of the peptide-containing acrylic resin is preferably 50,000 or more, more preferably 100,000 or more, preferably 1,000,000 or less, more preferably 800,000 or less. When the weight-average molecular weight is equal to or more than the lower limit and equal to or less than the upper limit, the effects of the present invention can be more effectively exhibited. When the weight-average molecular weight is equal to or less than the upper limit, the extensibility of cells during cell culture can be more effectively improved.

[0072] The weight average molecular weight of the peptide-containing acrylic resin can be measured, for example, by the following method. The peptide-containing acrylic resin is dissolved in tetrahydrofuran (THF) to prepare a 0.2% solution of the peptide-containing acrylic resin. Next, the weight average molecular weight is evaluated under the following measurement conditions using a gel permeation chromatography (GPC) measuring device (APC system, Waters).

[0073] Column: HSPgel HR MB-M 6.0 x 150 mm Flow rate: 0.5mL / min Column temperature: 40℃ Injection volume: 10μl Detector: RI, PDA Standard sample: polystyrene

[0074] (Scaffolding material for cell culture) The scaffold material is used for culturing cells. The scaffold material is used as a scaffold for cells when culturing the cells.

[0075] The above cells include animal cells such as human, mouse, rat, pig, cow, and monkey cells. The above cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The above somatic cells may be cancer cells.

[0076] The above stem cells include mesenchymal stem cells (MSC), iPS cells, ES cells, and Muse cells. , embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0077] Examples of the mature cells include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

[0078] The above-mentioned scaffold material is preferably used for two-dimensional culture (plate culture), three-dimensional culture or suspension culture of cells, and more preferably for two-dimensional culture (plate culture).

[0079] The scaffold material is preferably used for serum-free medium culture. Since the scaffold material contains the peptide-containing acrylic resin, it can enhance the adhesiveness of cells even in serum-free medium culture that does not contain feeder cells or adhesive proteins, and in particular, can further enhance the initial fixation rate after cell seeding.

[0080] In the scaffold material (100% by weight), the content of the peptide-containing acrylic 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). Therefore, the scaffold material most preferably contains only the peptide-containing acrylic resin. When the content of the peptide-containing acrylic resin is equal to or more than the lower limit, the effect of the present invention can be more effectively exhibited.

[0081] The scaffold material may contain components other than the peptide-containing acrylic resin, 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.

[0082] From the viewpoint of effectively exerting the effects of the present invention, the content of components other than the peptide-containing acrylic resin is preferably as small as possible. In 100% by weight of the scaffold material, the content of the components is preferably 10% by weight or less, more preferably 5% by weight or less, even more preferably 2.5% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight (not contained). Therefore, it is most preferable that the scaffold material does not contain any components other than the peptide-containing acrylic resin.

[0083] It is preferable that the scaffold material is substantially free of animal-derived raw materials. By not containing any animal-derived raw materials, it is possible to provide a scaffold material for cell culture that is highly safe and has little variation in quality during production. Here, "substantially free of animal-derived raw materials" means that the amount of animal-derived raw materials in the scaffold material is 3% by weight or less. In the scaffold material, it is preferable that the amount 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.

[0084] The shape of the scaffold material is not particularly limited. The scaffold material may be particles, fibers, a porous body, or a film. The particles, fibers, porous body, and film may each contain components other than the scaffold material.

[0085] The scaffold material can also be used as a cell culture carrier (medium) containing the scaffold material and a polysaccharide. The polysaccharide is not particularly limited, and any conventionally known polysaccharide can be used. The polysaccharide is preferably a water-soluble polysaccharide.

[0086] The scaffold material can also be used as a fiber for cell culture comprising a fiber body and a scaffold material disposed on the surface of the fiber body. In this case, the scaffold material for cell culture is a fiber It is preferable that the fiber is coated on the surface of the main body, and is preferably a coating. In this fiber for cell culture, a scaffold material may be present in the fiber body. For example, the fiber body can be impregnated with or kneaded with a liquid scaffold material to allow the scaffold material to be present in the fiber body. Stem cells generally have a property of being difficult to adhere to planar structures and easy to adhere to three-dimensional structures such as fibrous structures, so that the fiber for cell culture is preferably used for three-dimensional culture of stem cells. Among stem cells, the fiber for cell culture is more preferably used for three-dimensional culture of adipose stem cells.

[0087] <Resin film> The resin film according to the present invention is a resin film formed by the above-mentioned scaffold material. The resin film is formed by using a scaffold material for cell culture. The resin film is preferably a membranous scaffold material. The resin film is preferably a membranous scaffold material.

[0088] The thickness of the resin film is not particularly limited. The average thickness of the resin film may be 50 nm or more, 500 nm or more, 1000 μm or less, or 500 μm or less.

[0089] The resin film is immersed in 37°C ion-exchanged water for 24 hours, and then the compressive modulus at a frequency of 1 Hz measured in the ion-exchanged water using a nanoindenter device in accordance with ISO14577-1 is preferably 1 GPa or more, more preferably 1.5 GPa or more, and even more preferably 2 GPa or more. When the compressive modulus is equal to or more than the lower limit, the extensibility of cells during cell culture can be improved. The upper limit of the compressive modulus is not particularly limited. The compressive modulus may be 15 GPa or less.

[0090] The compressive elastic modulus is measured as follows.

[0091] The resin film is placed in a beaker filled with ion-exchanged water, and the beaker is left in a thermostatic chamber at 37° C. for 24 hours. The resin film that has been immersed in water for 24 hours is measured in ion-exchanged water using a nanoindenter (e.g., Triboindenter, manufactured by Hysitron) at a frequency of 1 Hz in accordance with ISO14577-1. The compressive elastic modulus is calculated according to the following formula.

[0092] Compressive elastic modulus = √π × (slope of load-displacement curve in elastic region) / (2 × √(contact projected area))

[0093] Here, the elastic region refers to the region where the slope of the load-displacement curve is constant, and the contact projection area refers to the area where the indenter and the sample are in contact.

[0094] The indenter used is a Berkovich (triangular pyramid type, tip diameter R number 100 nm), and the indentation depth can be set to 50 nm.

[0095] The compressive modulus can be well adjusted, for example, by adjusting the type and weight-average molecular weight of the peptide-containing acrylic resin. For example, by reducing the SP value of the peptide-containing acrylic resin, increasing the weight-average molecular weight, or forming a crosslinked structure between molecules, swelling in water can be suppressed and the compressive modulus can be increased.

[0096] The water swelling ratio of the resin film is preferably 70% or less, more preferably 50% or less, even more preferably 40% or less, even more preferably 30% or less, and particularly preferably 20% or less. When the water swelling ratio is equal to or less than the upper limit, the effect of the present invention can be more effectively exhibited. The lower limit of the water swelling ratio of the resin film is not particularly limited. The water swelling ratio of the resin film may be 0.5% or more.

[0097] The water swelling ratio is measured as follows.

[0098] The resin film is cut out to obtain a resin film (test piece) of 1 cm length x 3 cm width x 100 μm thickness. The scaffold material may be molded into a membrane to obtain a test piece of the above size. The obtained test piece is immersed in 50 g of pure water at 37° C. for 24 hours, and the immersed test piece is filtered through a plain woven wire mesh with a mesh size of 200 mesh to obtain a swollen test piece. The water swelling ratio is calculated according to the following formula.

[0099] Water swelling ratio (%) = (weight of swollen test piece (g) - weight of test piece (g)) / (weight of test piece (g)) x 100

[0100] From the viewpoint of further enhancing the adhesiveness and proliferation of cells, the resin film preferably has a phase-separated structure having at least a first phase and a second phase.

[0101] Examples of the phase separation structure include microphase separation structures such as a sea-island structure, a cylindrical structure, a gyroid structure, and a lamellar structure. In the sea-island structure, for example, the first phase can be the sea portion, and the second phase can be the island portion. In the cylindrical structure, the gyroid structure, or the lamellar structure, for example, the phase with the largest surface area can be the first phase, and the phase with the second largest surface area can be the second phase. The resin film has a continuous phase and a discontinuous phase, which can increase the affinity with cells and further increase the adhesiveness and proliferation of cells.

[0102] The phase separation structure is preferably a sea-island structure. The resin film preferably has a sea-island structure. In this case, the adhesiveness and proliferation of cells can be further improved.

[0103] When the resin film has an island-in-a-sea structure, the surface area ratio of the islands (second phase) to the entire surface of the resin film is preferably 0.01 or more, more preferably 0.1 or more, even more preferably 0.2 or more, and is preferably 0.95 or less, more preferably 0.9 or less, even more preferably 0.8 or less. When the surface area ratio is equal to or more than the lower limit and equal to or less than the upper limit, the adhesiveness of cells can be further increased.

[0104] When the resin film has a sea-island structure, it is preferable that the island portion contains a peptide portion. That is, it is preferable that the resin film has a sea portion and an island portion, and the island portion contains a peptide portion. In this case, the adhesion domain of the cell accumulates in the island portion, thereby further enhancing the adhesiveness of the cell.

[0105] The presence or absence of a phase-separated structure can be confirmed by, for example, an atomic force microscope (AFM), a transmission electron microscope (TEM), a scanning electron microscope (SEM), etc. The surface area ratio can be determined from a microscope observation image by using image analysis software such as ImageJ.

[0106] The phase-separated structure can be formed, for example, by increasing the content of peptide moieties and forming an inter- or intramolecular phase-separated structure in the peptide-containing polyvinyl alcohol derivative.

[0107] (Cell culture container) The cell culture vessel 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 a surface of the vessel body.

[0108] FIG. 1 is a cross-sectional view that illustrates a cell culture vessel according to one embodiment of the present invention.

[0109] 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.

[0110] 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, the resin film may be disposed on the surface of the second container body.

[0111] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0112] The container body may be a cell culture plate or a cell culture flask having one or more wells (holes). The number of wells in the plate is not particularly limited. The number of wells may be, but is not particularly limited to, 2, 4, 6, 12, 24, 48, 96, 384, etc. The shape of the well may be, but is not particularly limited to, a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom of the well may be, but is not particularly limited to, a flat bottom, a round bottom, an uneven bottom, etc.

[0113] The material of the container body is not particularly limited, but examples thereof include resins, metals, and inorganic materials. Examples of the resins include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimide, polyamide, polyamideimide, (meth)acrylic resins, epoxy resins, and silicone. Examples of the metals include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, and platinum. Examples of the inorganic materials include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, and silicon nitride.

[0114] (Cell culture method) Cells can be cultured using the scaffold material and the resin film. 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. Examples of the cells include the cells described above.

[0115] The cell culture method preferably includes a step of seeding cells on the scaffold material. The cell culture method preferably includes a step of seeding cells on the resin film. The cells may be cell clusters. The cell clusters can be obtained by adding a cell release agent to a confluent culture vessel and homogenously disrupting the cells by pipetting. The cell release agent is not particularly limited, but is preferably an ethylenediamine / phosphate buffer solution. The size of the cell clusters is preferably 50 μm to 200 μm.

[0116] 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.

[0117] The content of structural units in the obtained peptide-containing acrylic resin was measured by 1H-NMR (nuclear magnetic resonance spectroscopy) after dissolving the synthetic resin in DMSO-d6 (dimethyl sulfoxide). , measured by FT-IR or LC-MS.

[0118] Example 1 Preparation of peptide-containing acrylic resin: An acrylic monomer solution was obtained by dissolving 29 parts by weight of butyl acrylate, 3 parts by weight of 2-hydroxyethyl acrylate, and 1 part by weight of acrylic acid in 30 parts by weight of tetrahydrofuran. 1 part by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained acrylic monomer solution, and the obtained liquid was applied onto a PET film. The applied 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 Eye Graphics Co., Ltd.) at 25°C. 2 The acrylic resin solution was obtained by irradiating the acrylic resin with light at 400 nm for 3 hours. The acrylic resin solution was then vacuum dried at 80° C. for 3 hours to obtain an acrylic resin.

[0119] The weight-average molecular weight of the resulting acrylic resin was about 100,000.

[0120] The obtained acrylic resin was dissolved in butanol solution to obtain an acrylic resin-containing butanol solution at 3% by weight. 150 μL of the obtained acrylic resin-containing butanol solution was dispensed onto a φ22 mm cover glass (Matsunami Co., Ltd., 22 round No. 1, which was used after removing dust with an air duster), and the glass was rotated at 2000 rpm for 20 seconds using a spin coater to obtain a smooth resin film.

[0121] Next, a linear peptide having an amino acid sequence of Gly-Arg-Gly-Asp-Ser (5 amino acid residues, indicated as GRGDS in the table) was prepared. 1 part by weight of this peptide and 1 part by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to calcium- and magnesium-free phosphate buffered saline so that the final concentration of the peptide was 1 mM to prepare a peptide-containing liquid. 1 part by weight of this peptide-containing liquid was added to the obtained resin film to dehydrate and condense the carboxyl group of the acrylic resin and the amino group of Arg of the peptide. In this way, the peptide-containing acrylic resin (resin film) of Example 1 was obtained.

[0122] Preparation of cell culture vessels: The resulting laminate of the peptide-containing acrylic resin (resin film) and cover glass was placed in a polystyrene dish having a diameter of 22 mm to obtain a cell culture vessel.

[0123] (Examples 2 and 3) A peptide-containing acrylic resin (resin film) having the configuration shown in Table 1 was obtained in the same manner as in Example 1, except that the blending amounts of butyl acrylate and 2-hydroxyethyl acrylate were changed. In addition, a cell culture vessel was obtained in the same manner as in Example 1, except that the obtained peptide-containing acrylic resin was used.

[0124] Comparative Example 1 A peptide-containing acrylic resin (resin film) having the configuration shown in Table 2 was obtained in the same manner as in Example 1, except that methoxy-triethylene glycol acrylate was used instead of butyl acrylate and 2-hydroxyethyl acrylate. In addition, a cell culture vessel was obtained in the same manner as in Example 1, except that the obtained peptide-containing acrylic resin was used.

[0125] Comparative Example 2 A peptide-containing acrylic resin (resin film) having the configuration shown in Table 2 was obtained in the same manner as in Example 1, except that 2-hydroxyethyl acrylate was not used and the blending amount of butyl acrylate was changed. In addition, a cell culture vessel was obtained in the same manner as in Example 1, except that the obtained peptide-containing acrylic resin was used.

[0126] Comparative Example 3 An acrylic resin (resin film) having the configuration shown in Table 2 was obtained in the same manner as in Example 1, except that the blending amounts of butyl acrylate and 2-hydroxyethyl acrylate were changed and that peptide and acrylic acid were not used. In addition, a cell culture vessel was obtained in the same manner as in Example 1, except that the obtained acrylic resin was used.

[0127] (evaluation) (1) Solubility parameter (SP value) of the first structural part in the peptide-containing acrylic resin The solubility parameter (SP value) of the first structural portion in the peptide-containing acrylic resin was calculated by Okitsu's formula.

[0128] (2) Compressive elastic modulus of resin membrane in ion-exchanged water A cell culture vessel having a resin film formed from a scaffold material for cell culture was placed in a beaker filled with ion-exchanged water, and the beaker was left in a thermostatic bath at 37°C for 24 hours. The cell culture vessel that had been submerged in water for 24 hours was taken out with the resin film submerged in the ion-exchanged water. The compressive elastic modulus of the resin film was measured in ion-exchanged water at a frequency of 1 Hz using a nanoindenter device (Triboindenter, manufactured by Hysitron) in accordance with ISO14577-1. The compressive elastic modulus was calculated according to the following formula.

[0129] Compressive elastic modulus = √π × (slope of load-displacement curve in elastic region) / (2 × √(contact projected area))

[0130] The elastic region refers to the region where the slope of the load-displacement curve is constant, and the contact projection area refers to the area where the indenter and the sample are in contact.

[0131] The indenter used was a Berkovich (triangular pyramid type, tip diameter R number 100 nm), and the indentation depth was set to 50 nm.

[0132] (3) Water swelling ratio of resin film The obtained resin film was cut out to obtain a resin film (test piece) with a length of 1 cm, a width of 3 cm, and a thickness of 100 μm. The obtained test piece was immersed in 50 g of pure water at 37° C. for 24 hours, and the immersed test piece was filtered using a plain woven wire mesh with an opening of 200 mesh to obtain a swelling test piece. The water swelling ratio was calculated according to the following formula.

[0133] Water swelling ratio (%) = (weight of swollen test piece (g) - weight of test piece (g)) / (weight of test piece (g)) x 100

[0134] (4) Presence or absence of an island structure The obtained resin film was immersed in a PBS solution for 30 minutes. After immersion, the resin film was observed using an atomic force microscope (AFM, Bruker's "Dimension XR"). The measurement conditions were set to a peak set point of 2 nN in QNM mode, and an area of ​​1 μm x 1 μm was observed. The obtained height mapping image was compared with the elastic modulus mapping image to determine the presence or absence of a sea-island structure.

[0135] (5) Cell culture evaluation (cell adhesion) The following liquid medium and ROCK (Rho-associated kinase) specific inhibitor were prepared.

[0136] TeSR E8 medium (STEM CELL) ROCK Inhibitor (Y27632)

[0137] 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.

[0138] A colony of h-iPS cells 253G1 that had reached a confluent state was placed in a φ35 mm dish, 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution was added, and the dish was left to stand at room temperature for 2 minutes. After removing the ethylenediamine / phosphate buffer solution, cell clusters were obtained that were broken into pieces of 50 μm to 200 μm in size by pipetting with 1 mL of TeSR E8 medium. The resulting cell clusters (cell count 0.2 × 10 5 The cells were clamp-seeded onto the resin membrane of a cell culture vessel.

[0139] At the time of seeding, 1.5 mL of liquid medium and a ROCK-specific inhibitor were added to the cell culture vessel to a final concentration of 10 μM, and the cells were cultured in an incubator at 37°C with a CO2 concentration of 5%. After that, the liquid in the cell culture vessel was replaced with 1.5 mL of fresh liquid medium every 24 hours, and the cells were cultured for 5 days. During this time, cells that were free or floating from the resin membrane were collected by pipetting and discarded.

[0140] After 5 days of culture, the entire resin film was photographed using a phase-contrast microscope, and the area of ​​the cell masses relative to the area of ​​the resin film was calculated. The cell adhesion was evaluated according to the following criteria.

[0141] <Criteria for evaluation of cell culture (cell adhesion)> AA: Cell mass occupies 70% or more of the surface area A: The area occupied by cell aggregates is 50% or more and less than 70% B: The area occupied by cell aggregates is less than 50%

[0142] Details and results are shown in Tables 1 and 2 below.

[0143] [Table 1]

[0144] [Table 2] [Explanation of symbols]

[0145] 1... Container for cell culture 2... Container body 2a... Surface 3... Resin film

Claims

1. A peptide-containing acrylic resin is included. the peptide-containing acrylic resin has a first structural portion that does not have a peptide moiety in a side chain and a second structural portion that has a peptide moiety in a side chain, the first structural portion has a structural unit derived from a (meth)acrylic acid ester, an average ratio of structural units derived from the (meth)acrylic acid ester in 100 mol % of all structural units contained in the first structural portion is 80 mol % or more; The solubility parameter of the first structural portion calculated by the Okitsu formula is 9.8 (cal / cm ３ ) １／２ Above, 10.5 (cal / cm ３ ) １／２ is as follows: A scaffold material for cell culture, wherein the peptide portion in the second structural portion has a cell adhesive amino acid sequence.

2. A scaffold material for cell culture as described in claim 1, wherein the (meth)acrylic acid ester includes a (meth)acrylic acid alkyl ester.

3. A scaffold material for cell culture as described in claim 1 or 2, wherein the cell adhesive amino acid sequence has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence.

4. A scaffold material for cell culture described in any one of claims 1 to 3, wherein the cell adhesive amino acid sequence has at least an RGD sequence represented by the following formula (1): Arg-Gly-Asp-X...Formula (1) In the formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

5. The cell culture scaffold material according to any one of claims 1 to 4, wherein the number of amino acid residues in the peptide portion in the second structural portion is 10 or less.

6. A resin film formed from the scaffold material for cell culture according to any one of claims 1 to 5.

7. The resin film according to claim 6, which has a compressive elastic modulus of 1 GPa or more at a frequency of 1 Hz measured in ion-exchanged water at 37°C in accordance with ISO14577-1 using a nanoindenter device after immersion in the ion-exchanged water for 24 hours.

8. The resin film according to claim 6 or 7, which has a water swelling ratio of 70% or less.

9. It has an island structure. The resin film according to any one of claims 6 to 8, wherein an island portion in the sea-island structure contains the peptide portion.

10. A cell culture vessel comprising the resin film according to any one of claims 6 to 9 in at least a part of a cell culture region.

11. A method for culturing cells, comprising the step of seeding cells on the resin film according to any one of claims 6 to 9.

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