Cell cultivation or proliferation substrate

Abstract

Provided is a cell cultivation or proliferation substrate that enables more convenient separation of cells without using chemicals. A cell cultivation or proliferation substrate that comprises a gel-type or gel-forming sol-type composition, or a dried product of the composition, containing a polypeptide having an elastin-like block peptide sequence containing a G sequence block of (SEQ ID NO: 1: X1GGX2G)n (in the formula: X1s are the same or different and represent V or L; X2s are the same or different and represent V or L; and n is an integer of 4 or more) and a P sequence block of (SEQ ID NO:2: VPGX3G)m (in the formula: X3s are the same or different and represent an arbitrary amino acid; and m is an integer of 5 or more).

Description

Cell culture or growth substrate

[0001] The present invention relates to a cell culture or proliferation substrate and the like.

[0002] Three-dimensional cell culture is a method of culturing cells in an artificially created three-dimensional environment. Three-dimensional cell culture allows for the formation of three-dimensional cell arrangements and interactions, and compared to two-dimensional cell culture, it can mimic an in vivo environment more closely. Therefore, three-dimensional cell culture has attracted particular attention in a wide range of fields, including regenerative medicine, tumor biology, and drug discovery research, as a method for reproducing the high functionality of living tissue. Furthermore, in recent years, cell sheets created from two-dimensional cell culture have been used in regenerative medicine.

[0003] In three-dimensional cell culture, extracellular matrix gels such as collagen are used as cell culture or growth substrates. However, enzymatic digestion or other methods are required to separate cells from the substrate after culture. Similarly, enzymatic digestion or other methods are required to separate cells from the substrate after culture in two-dimensional cell culture.

[0004] Elastin is an important functional protein that provides elasticity and stretchability to biological tissues. However, its highly hydrophobic nature makes it difficult to handle, significantly slowing down its use as a material. The present inventors have developed a doubly hydrophobic elastin-like polypeptide (GPG) that forms self-assembling nanofibers in water (Non-Patent Document 1). The resulting fibers can form physical gels, which are promising for application as biomaterials.

[0005] DHT Le, R. Hanamura, D.-H. Pham, M. Kato, DA Tirrell, T. Okubo, A. Sugawara-Narutaki, Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides, Biomacromolecules. 14 (2013) 1028-1034. https: / / doi.org / 10.1021 / bm301887m.

[0006] An object of the present invention is to provide a cell culture or proliferation substrate that allows cells to be separated more easily and without the use of chemicals.

[0007] In view of the above problems, the present inventors have conducted extensive research and have found that (SEQ ID NO: 1:X 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 are the same or different and represent any amino acid, and m is an integer of 5 or greater.) or a cell culture or proliferation substrate comprising a dried product of the composition, the present inventors have found that the above-mentioned problems can be solved by a gel-like or gel-forming sol-like composition containing a polypeptide comprising an elastin-like block peptide sequence comprising the elastin-like block peptide sequence, or a cell culture or proliferation substrate comprising the dried product of the composition. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. (SEQ ID NO: 1:X 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 are the same or different and represent any amino acid, and m represents an integer of 5 or more.) A gel-like or gel-forming sol-like composition containing a polypeptide comprising an elastin-like block peptide sequence comprising the elastin-like block peptide sequence, or a cell culture or growth substrate comprising a dried product of said composition.

[0009] Item 2. The cell culture or proliferation substrate according to Item 1, wherein the sequence structure of the elastin-like block peptide sequence is, from the N-terminus, G sequence block-P sequence block-G sequence block, G sequence block-P sequence block-P sequence block-G sequence block, P sequence block-G sequence block, or G sequence block-P sequence block.

[0010] Item 3. The cell culture or proliferation substrate according to Item 1 or 2, for use in three-dimensional culture or three-dimensional proliferation of cells.

[0011] Item 4. The cell culture or proliferation substrate according to Item 1 or 2, for use in two-dimensional culture or two-dimensional proliferation of cells.

[0012] Item 5. The cell culture or proliferation substrate according to any one of Items 1 to 4, wherein the composition retains cells.

[0013] Item 6. The cell culture or proliferation substrate according to any one of Items 1 to 5, wherein the concentration of the polypeptide in the composition is 0.2 w / v % or more.

[0014] Item 7. The cell culture or proliferation substrate according to any one of Items 1 to 6, wherein the concentration of the polypeptide in the composition is 0.2 to 3 w / v %.

[0015] Item 8. The cell culture or proliferation substrate according to any one of Items 1 to 7, for use by converting the gel-like composition into a sol and / or for use by converting the sol-like composition into a gel.

[0016] Item 9. A method for culturing or growing cells, comprising culturing or growing cells on the cell culture or growth substrate according to any one of Items 1 to 8.

[0017] Item 10. A cell population obtainable by the cell culture or proliferation method according to Item 9.

[0018] According to the present invention, it is possible to provide a cell culture or proliferation substrate that allows cells to be separated more easily and without using chemicals.

[0019] TEM observation image of the GPG1 gel obtained in Test Example 2. A photograph of the tube containing the gel is shown in the upper right of the image. Rheology measurement results of Test Example 3 are shown. The vertical axis shows the storage modulus (G') and loss modulus (G"). The horizontal axis shows the elapsed time of the test (unit: s (seconds)). Rheology measurement results at the time of initial gelation in Test Example 4 are shown. A: Time dispersion measurement (constant frequency of 1 Hz, constant strain of 1%), B: Strain dispersion measurement (constant frequency of 1 Hz), C: Frequency dispersion measurement (constant strain of 1%). Rheology measurement results at the time of regelation (no cells) in Test Example 5 are shown. A: Time dispersion measurement (constant frequency of 1 Hz, constant strain of 1%), B: Strain dispersion measurement (constant frequency of 1 Hz), C: Frequency dispersion measurement (constant strain of 1%). Rheology measurement results at the time of regelation (with cells) in Test Example 6 are shown. A: Time dispersion measurement (constant frequency of 1 Hz, constant strain of 1%), B: Strain dispersion measurement (constant frequency of 1 Hz), C: Frequency dispersion measurement (Constant strain of 1%). Microscopic images of Test Example 7 are shown. "In gel" indicates an image of cells in the gel, and "Recovery" indicates an image of cells dispersed in PBS after solation. Phase-contrast microscope images are shown 7 days after the start of differentiation induction in Test Example 9. Immunostained images of the gel 7 days after the start of differentiation induction in Test Example 9 are shown. A indicates an α-actinin stained image, B indicates a cell nucleus stained image, and C indicates a merged image of A and B.

[0020] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0021] In this specification, amino acid residues in an amino acid sequence may be simply represented by the amino acid or by a specific amino acid name (valine, leucine), etc. In addition, amino acid residues in an amino acid sequence may be represented by a single-letter code for the amino acid.

[0022] In one aspect, the present invention provides a method for the preparation of a nucleic acid sequence comprising the steps of: 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 The present invention relates to a gel-like or gel-forming sol-like composition (sometimes referred to herein as the "composition of the present invention") containing a polypeptide (sometimes referred to herein as the "polypeptide of the present invention") comprising an elastin-like block peptide sequence comprising an elastin-like block peptide sequence comprising an elastin-like block peptide sequence comprising an amino acid sequence selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104,

[0023] The G sequence block is an amino acid sequence shown in SEQ ID NO: 1 (X 1 GGX 2 G) repeat sequence (X 1 GGX 2 G) n There are no particular limitations on the G sequence block, as long as it is a block consisting of the following. The G sequence block is usually a block that can form a β-sheet structure. The G sequence block can impart the ability to form fibers by self-assembly to the polypeptide.

[0024] X 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L. X 1 and X 2 is preferably V.

[0025] n represents an integer of 4 or greater. By setting n to 4 or greater, the polypeptide of the present invention can form fibrous self-assemblies. n is preferably 4 to 20, more preferably 4 to 12, even more preferably 4 to 8, and even more preferably 4 to 6.

[0026] The P sequence block is the amino acid sequence shown in SEQ ID NO: 2 (VPGX 3 G) repeat sequence (VPGX 3 G) mThere are no particular limitations as long as it is a block consisting of the P sequence block. The P sequence block is usually a block that can form a β-turn structure. The P sequence block gives the polypeptide a lower critical solution temperature (LCST), and it self-assembles and phase-separates from an aqueous solution above the LCST.

[0027] The LCST of the polypeptide of the present invention (solvent: water, concentration: 0.03 wt%) is, for example, 10 to 30°C, preferably 15 to 25°C.

[0028] X 3 X represents any amino acid, whether identical or different. 3 Examples of the amino acid represented by the formula (X) include amino acids having a basic side chain such as lysine, arginine, and histidine; amino acids having an acidic side chain such as aspartic acid and glutamic acid; amino acids having an uncharged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids having a nonpolar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having a β-branched side chain such as threonine, valine, and isoleucine; and amino acids having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, and histidine. 3 The amino acid represented by is preferably an amino acid other than proline.

[0029] X 3 The LCST can be adjusted by the amino acid represented by X. 3 If the amino acid represented by X in the P sequence block is a hydrophilic amino acid, the LCST becomes too high, which is undesirable. From the viewpoint of easily adjusting the LCST to an appropriate (physiologically acceptable) temperature (e.g., 30 to 40°C), 3 It is preferred that some of the amino acids represented by are non-aromatic hydrophobic amino acids such as valine and leucine (preferably valine), and other amino acids are aromatic hydrophobic amino acids such as phenylalanine and tryptophan (preferably phenylalanine). In this case, X in the P sequence block 3 X to the total number of 3The ratio of the number of non-aromatic hydrophobic amino acids represented by X in the P sequence block is, for example, 60 to 95%, preferably 70 to 80%, more preferably 75 to 85%. 3 X to the total number of 3 The ratio of the number of aromatic hydrophobic amino acids represented by X in the P sequence block is, for example, 5 to 40%, preferably 20 to 30%, more preferably 15 to 25%. 3 It is preferable that the aromatic hydrophobic amino acids represented by the formula (VPGX) are distributed as widely as possible, for example, 1 to 7, preferably 1 to 5, more preferably 2 to 4, and even more preferably 3 repeat units (VPGX 3 G) at a rate of one for each iteration, x 3 It is preferred that repeat units in which the amino acid is an aromatic hydrophobic amino acid occur.

[0030] m represents an integer of 5 or greater. m is preferably 10 to 100, more preferably 10 to 50, even more preferably 15 to 35, and particularly preferably 20 to 30.

[0031] The elastin-like block peptide sequence is not particularly limited as long as it contains a G sequence block and a P sequence block. The number of G sequence blocks in the elastin-like block peptide sequence is not particularly limited, but is, for example, 1 to 5, preferably 2 to 5, more preferably 2 to 4, even more preferably 2 to 3, and particularly preferably 2. The number of P sequence blocks in the elastin-like block peptide sequence is not particularly limited, but is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

[0032] The blocks (between a G sequence block and a P sequence block, between a G sequence block and a G sequence block, or between a P sequence block and a P sequence block) may be directly linked or may be linked via a linker sequence. Preferably, a linker sequence is interposed between the blocks.

[0033] The linker sequence is not particularly limited as long as it does not significantly reduce the fiber-forming ability of the elastin-like block peptide sequence due to its self-assembly, and generally, any amino acid or amino acid sequence can be used without major limitations. The linker sequence may have a length of, for example, 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids, even more preferably 1 to 8 amino acids, and even more preferably 2 to 8 amino acids. Specific examples of linker sequences include the amino acid sequence represented by SEQ ID NO: 7: LWLGSG and the amino acid sequence represented by KL. Furthermore, amino acid sequences obtained by mutation (e.g., substitution, deletion, insertion, addition, preferably substitution, more preferably conservative substitution) of one or more amino acids (e.g., 1 to 5 amino acids, preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids, and even more preferably 1 amino acid) of these amino acid sequences can also be used.

[0034] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with an amino acid having a similar side chain. For example, substitution between amino acids having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acids having acidic side chains such as aspartic acid and glutamic acid; amino acids having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having β-branched side chains such as threonine, valine, and isoleucine; and amino acids having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0035] From the viewpoint of fiber-forming ability through self-assembly, the sequence structure of the elastin-like block peptide sequence is preferably, from the N-terminus, G sequence block-P sequence block-G sequence block, G sequence block-P sequence block-P sequence block-G sequence block, P sequence block-G sequence block, or G sequence block-P sequence block. The sequence structure is particularly preferably G sequence block-P sequence block-G sequence block, or G sequence block-P sequence block-P sequence block-G sequence block. In these structures, "-" indicates a direct bond (between blocks) or another sequence (for example, a linker sequence, etc.).

[0036] The polypeptide of the present invention can have a cell adhesive sequence at its terminus. The cell adhesive sequence is not particularly limited, and various sequences can be used. The cell adhesive sequence can be one that does not have cell selectivity, such as RGDS (SEQ ID NO: 8), or one that has cell selectivity (e.g., REDV (SEQ ID NO: 20)). Sequences with cell selectivity include, for example, HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, G PS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GN Examples include S, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 9), LPGFPGTP (SEQ ID NO: 10), GPPGLSGPP (SEQ ID NO: 11), FPGPPGPP (SEQ ID NO: 12), LPGLPGPP (SEQ ID NO: 13), FPGLPGPP (SEQ ID NO: 14), GPPGPPGSPG (SEQ ID NO: 15), LPGPPGPP (SEQ ID NO: 16), FPGSPGFPG (SEQ ID NO: 17), GSPGLPGTP (SEQ ID NO: 18), and IGLSGEKG (SEQ ID NO: 19).

[0037] When the polypeptide of the present invention comprises a cell adhesion sequence, the cell adhesion sequence is located at the termini (N-terminus and / or C-terminus) of the polypeptide of the present invention, preferably at the C-terminus. The cell adhesion sequence may be located at either the N-terminus or the C-terminus, or at both. In a preferred embodiment of the present invention, the cell adhesion sequence is located at only one terminus.

[0038] When the polypeptide of the present invention contains a cell adhesive sequence, the elastin-like block peptide sequence and the cell adhesive sequence may be directly linked, or another amino acid sequence (e.g., a sequence of 3 to 30, 7 to 25, or 10 to 20 amino acids) may be interposed between them. From the viewpoint of cell adhesiveness, this other amino acid sequence is preferably a hydrophilic amino acid-rich sequence. In other words, it is preferable that the hydrophilic amino acid-rich sequence is located on the opposite side of the terminal end of the cell adhesive sequence. The hydrophilic amino acid-rich sequence is not particularly limited as long as it contains a high proportion of hydrophilic amino acids, and examples include amino acid sequences of 3 to 15 or 5 to 10 amino acids in length that contain hydrophilic amino acids at a ratio of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. A preferred hydrophilic amino acid-rich sequence is a histidine-stranded sequence (His tag sequence), which facilitates purification of the polypeptide of the present invention. The other amino acid sequence preferably contains an amino acid with a functional group that can be used in a crosslinking reaction (e.g., lysine, cysteine, etc.).

[0039] The polypeptide of the present invention may be chemically modified as long as its ability to form a thixotropic gel is not significantly impaired. The ability to form a thixotropic gel can be evaluated according to Test Examples 2 and 3 described below.

[0040] The polypeptide of the present invention has a C-terminus containing a carboxyl group (-COOH), a carboxylate group (-COO － ), amide (-CONH2) or ester (-COOR).

[0041] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0042] In the polypeptide of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0043] Furthermore, in the polypeptide of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1－6 C such as alkanoyl group 1－6 Also included are those protected by an acyl group or the like, or conjugated proteins such as so-called glycoproteins to which sugar chains are bound.

[0044] The polypeptides of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0045] The polypeptide of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0046] The polypeptides of the present invention can be easily produced by known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0047] The composition of the present invention contains the polypeptide of the present invention and is in the form of a gel or a gel-forming sol.

[0048] A gel composition containing the polypeptide of the present invention can be obtained by dissolving the polypeptide of the present invention in a solvent at a temperature below the LCST (e.g., 0-15°C, 2-10°C), and then heating the resulting solution to a temperature above the LCST (e.g., 30-50°C, 30-45°C, 30-40°C) and allowing it to stand for a certain period of time (e.g., 1-300 hours, preferably 8-120 hours, more preferably 12-80 hours). Water is used as the solvent, but a mixed solvent of water and an organic solvent can also be used.

[0049] The concentration of the polypeptide of the present invention in the composition of the present invention is, from the viewpoint of gel-forming properties, for example, 0.2 w / v% or more, preferably 0.3 w / v% or more, and more preferably 0.35 w / v% or more. From the viewpoint of facilitating the penetration of other components (e.g., components necessary or suitable for cell culture) into the gel composition of the present invention, the concentration is, for example, 3.0 w / v% or less, preferably 2.0 w / v% or less, more preferably 1.5 w / v% or less, even more preferably 1.0 w / v% or less, and even more preferably 0.8 w / v% or less.

[0050] A gel-forming sol composition containing a polypeptide of the present invention is, in other words, a sol composition with gel-forming ability. Because the gel composition of the present invention exhibits thixotropy, a sol composition (sol composition 1) can be obtained by applying mechanical strain to the gel composition of the present invention (e.g., pipetting, vortexing, etc.). Sol composition 1 gels when the mechanical strain is removed or weakened, and therefore can be said to have gel-forming ability. Furthermore, a sol composition (sol composition 2) can be obtained by dissolving the polypeptide of the present invention in a solvent at a concentration low enough to prevent gel formation. Sol composition 2 gels when the concentration of the polypeptide of the present invention increases due to solvent evaporation, etc., and therefore can be said to have gel-forming ability. Furthermore, a sol composition (sol composition 3) can be obtained by dissolving the polypeptide of the present invention in a solvent at a temperature low enough to prevent gel formation (below the LCST). Sol composition 3 gels when heated to a gel-forming temperature (above the LCST) and allowed to stand for a certain period of time, and therefore can be said to have gel-forming ability.

[0051] The composition of the present invention preferably retains cells. Retaining cells can significantly improve the elastic modulus of the gel. There are no particular limitations on the manner in which cells are retained, but it is preferable that the cells are suspended in the composition of the present invention.

[0052] The biological species from which the cells are derived is not particularly limited and includes, for example, bacteria such as Enterobacteriaceae, fungi such as yeast, animals, and plants. Examples of animals include various mammals, particularly humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer, as well as non-mammalian vertebrates and invertebrates. In a preferred embodiment of the present invention, the biological species from which the target cells are derived include mammals, Escherichia coli, yeast, Bacillus subtilis, zebrafish, medaka, and insects. The type of cell is also not particularly limited and includes, for example, cells derived from various tissues or with various properties, such as blood cells, hematopoietic stem and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, neurons, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0053] When the composition of the present invention retains cells, the cell concentration is preferably 1.0 × 10 5　 cells / mL or more, preferably 2.0 × 10 5 cells / mL or more, more preferably 5.0 × 10 5 cells / mL or more, and even more preferably 1.0 x 10 6 The upper limit of the cell concentration is, for example, 1.0 × 10 8 cells / mL, 1.0 × 10 7 cells / mL, or 5.0 x 10 6 The total number of cells / mL can be calculated.

[0054] The method for retaining cells in the composition of the present invention is not particularly limited. Cells can be easily retained in the composition of the present invention by suspending them in the sol-like composition of the present invention. The obtained sol-like composition can be gelled to allow three-dimensional cell culture.

[0055] The composition of the present invention may contain other components in addition to those described above. These components include, but are not limited to, components necessary or suitable for cell culture. Examples of other components include standard inorganic salts such as magnesium, calcium, potassium, zinc, and iron, buffers, sugars, vitamins, essential amino acids, and non-essential amino acids. Specific examples include Dulbecco's Modified Eagle's Medium (DMEM), Minimal essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, αMinimal essential Medium (αMEM), Glasgow's Minimal essential Medium (GMEM), and Iscove's Modified Dulbecco's Medium (IMDM). In addition to the above, other components include serum, serum replacement components, extracellular matrix, other protein components, polysaccharides, and the like.

[0056] The content of other components can be, for example, 50 parts by weight or less, preferably 30 parts by weight or less, more preferably 20 parts by weight or less, even more preferably 10 parts by weight or less, and even more preferably 5 parts by weight or less, per 100 parts by weight of the polypeptide of the present invention.

[0057] When the composition of the present invention is in a gel form, its shape is not particularly limited as long as it is suitable as a substrate for cell culture or proliferation, and the shape may be, for example, a cube, a rectangular parallelepiped, a pyramid, a column, or a composite shape consisting of two or more shapes.

[0058] The dried composition of the present invention is a composition of the present invention in which the moisture content has been reduced, and is not particularly limited thereto. The dried composition of the present invention may have a moisture content of, for example, 50% by weight or less, 30% by weight or less, 20% by weight or less, 10% by weight or less, or 5% by weight or less. The dried composition of the present invention can be used as a substrate for cell culture or proliferation by adding water as needed to increase the moisture content.

[0059] The substrate of the present invention may consist solely of the composition of the present invention, or it may be a composite of the composition of the present invention and another substance. The other substance may be, for example, a structure that can form part of a cell culture vessel. Examples of materials for the structure include glass, resins (such as polystyrene, polyethylene, polypropylene, poly(acrylic acid ester), poly(methacrylic acid ester), polyacrylamide, polyacrylonitrile, polyethylene terephthalate, poly(L-lactic acid), poly(glycolic acid), poly(ε-caprolactone), poly(ethylene glycol) and copolymers thereof), polypeptides (such as collagen, gelatin, casein, fibroin, keratin, laminin, integrin, fibronectin, vitronectin), polysaccharides (such as cellulose, hyaluronic acid, chondroitin sulfate, starch, chitin, chitosan), silica, silicon, and metals (such as gold, silver, copper, iron, zinc, aluminum, nickel, and alloys or oxides thereof).

[0060] The substrate of the present invention is intended for use in cell culture or proliferation. Specifically, the substrate of the present invention is used to retain cells in the composition of the present invention or to place cells on the surface of the composition of the present invention and then culture or proliferate the cells. From this perspective, in one aspect, the present invention relates to a cell culture or proliferation method that includes culturing or proliferating cells on the substrate of the present invention. The substrate of the present invention can also be suitably used as bioink.

[0061] Here, "culturing or growing" includes not only cell division and growth, cell differentiation, etc., but also the maintenance of cells in a viable state. Furthermore, cells refer not only to cells in an isolated state, but also to cells in cell populations such as tissues, cell clumps, spheroids, and organoids. From this perspective, in one aspect, the present invention relates to a cell population that can be obtained by the above-described cell culture or growth method.

[0062] Components necessary or suitable for cell culture or proliferation can be supplied to cells by incorporating them in the composition of the present invention in advance, or by immersing the composition of the present invention, on the surface of which the cells are held or on which the cells are arranged, in a culture medium. Because the composition of the present invention can maintain a gel state even at low concentrations of the polypeptide of the present invention, even in the latter method, nutrients can be permeated into the composition of the present invention.

[0063] In a preferred embodiment, the substrate of the present invention can be used for three-dimensional cell culture or three-dimensional cell growth. In three-dimensional culture or growth, cells are dispersed in, for example, a gel-like composition of the present invention and cultured or grown. In this case, the components (medium components) necessary for culture or growth can be supplied to the cells by contacting a solution containing the medium components (culture medium) with the gel-like composition of the present invention, or by using a gel-like composition of the present invention containing the medium components. This can result in, for example, the formation of spheroids. Spheroids can be obtained by culturing or growing the cells for, for example, 3 days or more, preferably 3 to 10 days. After culturing or growing the cells, mechanical strain can be applied to the gel-like composition of the present invention to cause a sol-gel transition, and the cells in the sol can be recovered.

[0064] In a preferred embodiment, the substrate of the present invention can be used for two-dimensional cell culture or two-dimensional cell growth. In two-dimensional culture or growth, cells are placed on the surface of, for example, a gel-like composition of the present invention and cultured or grown. This allows, for example, the formation of a cell sheet. After culture or growth, the cells or cell sheet can be detached by leaving the composition at a relatively low temperature (for example, 25°C or lower, preferably 22°C or lower). Alternatively, after culture or growth, mechanical strain can be applied to the gel-like composition of the present invention to cause a sol-gel transition, allowing the cells or cell sheet to be detached.

[0065] The substrate of the present invention can also be suitably used as a bioink for cellular 3D bioprinting. For example, by suspending cells in the sol-like composition of the present invention and spatially arranging the cells, the desired three-dimensional tissue structure can be obtained through sol-gel transition.

[0066] The substrate of the present invention can be used not only as a cell culture substrate but also as a cell growth substrate for forming artificial blood vessels, a cell growth substrate for wound treatment, and the like.

[0067] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0068] Test Example 1. Synthesis of Polypeptides The following two polypeptides (GPG1 and GPG3): (GPG1) A G sequence block consisting of (SEQ ID NO: 3: VGGVG)5 from the N-terminus, (SEQ ID NO: 4: VPGXG) 25 We synthesized a polypeptide (SEQ ID NO: 5) containing an elastin-like block peptide sequence in which a P sequence block consisting of (wherein X is the same or different and represents V or F) and a G sequence block consisting of (SEQ ID NO: 3: VGGVG)5 are linked via a linker sequence, and a polypeptide (GPG3) in which a cell adhesion sequence is located at the C-terminus of GPG1 (SEQ ID NO: 6). GPG1 and GPG3 are polypeptides with previously reported amino acid sequences.

[0069] Specifically, E. coli BLR(DE3) strain was transformed with plasmid DNA encoding these polypeptides, and the polypeptides were expressed. The polypeptides were purified by metal ion affinity chromatography using a His-tag. The desired polypeptides were confirmed by SDS-PAGE and MALDI-TOF-MS.

[0070] The recovered peptide solution was placed in a Spectra / Por® 1 Dialysis Membrane MWCO: 6-8,000 (Spectrum Laboratories) and dialyzed against 2 L of ultrapure water at 4°C. The ultrapure water was changed twice every hour, then twice every two hours, and once every 1.5 hours, for a total of five dialysis sessions. After dialysis, the peptide solution was filtered through a Minisart® Syringe Filter 0.2 μm (SartoriusStedim Biotech) attached to the syringe to remove insolubilized peptides, and then frozen overnight at -80°C. The resulting solution was then lyophilized using a freeze dryer FDU-1200 (TOKYO RIKAKIKAI) to obtain powders of the two peptides, GPG1 and GPG3.

[0071] Test Example 2. Gelation of Polypeptide Solution GPG1 was dissolved in a 10 wt% aqueous sucrose solution to a final concentration of 0.5 wt%. The resulting GPG1 solution was heated to 37°C and left to stand. After one day, the GPG1 solution had turned into a transparent gel. TEM observation of the resulting gel confirmed the formation of nanofibers with diameters of 10 nm or less (Figure 1).

[0072] The same test was also performed on GPG3, and the same results as those for GPG1 were obtained.

[0073] Test Example 3: Thixotropy of Gel Rheology measurements were performed on the GPG1 gel obtained in Test Example 2 at a constant frequency of 1 Hz, alternating between 100% and 0.5% shear strain. The measurements were performed using a Modular Compact Rheometer MCR302 (Anton Paar) with a 1° cone and plate (25 mm diameter) and a 0.048 mm gap. To prevent the sample from drying during all measurements, Prowipes soaked in ultrapure water were placed around the measurement stage and covered with a solvent trap cover. The temperature was maintained at 37°C using the Peltier system built into the rheometer. An appropriate amount of each sample was placed on the measurement stage with a spatula, and the jig was lowered to the desired gap. The storage modulus (G') and loss modulus (G") were measured.

[0074] The results are shown in Figure 2. It was found that the GPG1 gel can repeatedly undergo reversible destruction under mechanical strain and recovery under static conditions, i.e., it exhibits thixotropy (self-repairing properties).

[0075] The same test was also performed on GPG3 gel, and the same results as those for GPG1 gel were obtained.

[0076] Test Example 4 Rheology at Initial Gelation GPG1 was dissolved in a 10 w / v % aqueous sucrose solution to a final concentration of 0.4 w / v %, and the resulting GPG1 solution was used as a sample.

[0077] The rheology measurement method was the same as in Test Example 3. A sample in solution at 4°C was placed in a rheometer MCR302 adjusted to 37°C, and dynamic viscoelasticity measurements were performed. The dynamic viscoelasticity measurements included three parameters: time dispersion, frequency dispersion, and strain dispersion. The storage modulus (G') and loss modulus (G") were measured for each parameter. First, a time dispersion measurement was performed for each sample, with measurements taken at a constant strain of 1% and a constant frequency of 1 Hz, with one point measured every 30 seconds for a total of 120 points, for a total of 60 minutes. The subsequent frequency dispersion measurement was performed at a constant strain of 1% over an angular frequency range of 100-0.1 rad / s. Finally, a strain dispersion measurement was performed at a constant frequency of 1 Hz over a shear strain range of 0.1-100%. For all samples, the strain dispersion measurement results indicated that the 1% strain was in the linear region.

[0078] The results are shown in Figure 3. From the time dispersion measurement in Figure 3A, at the first point immediately after the start of the measurement (30 seconds after the start of the measurement), G' was 28 Pa and G" was 56 Pa, with G" exceeding G'. With time, G' gradually increased and G" gradually decreased, until at 300 seconds, G' exceeded G". G' continued to increase, and after approximately 1000 seconds, its increase slowed down from around 1000 Pa, reaching approximately 6000 Pa by the end of the measurement. G" also exhibited a similar behavior to G', eventually reaching approximately 2000 Pa. In the strain dispersion measurement in Figure 3B, G' gradually decreased from 0.1% strain to approximately 1.46% strain, then rapidly decreased above 1.46% strain. On the other hand, G" tended to increase from 0.1% strain to approximately 0.5% strain, and then began to decrease above 0.5% strain, although the decrease was more gradual than G'. From 0.1% to approximately 7% strain, G' exceeded G", indicating elastic behavior. However, after approximately 7% strain, G" exceeded G', indicating viscous behavior. Frequency dispersion measurements shown in Figure 3C indicated that G' exceeded G" in the entire measured frequency range of 0.1-100 rad / s. Both G' and G" were within 10 3 The transition occurred on a Pa scale, with each value gradually increasing from the low frequency region to the high frequency region. Furthermore, the loss tangent tanδ, defined as tanδ (= G" / G'), was between 0.330 and 0.390.

[0079] The same test was also performed on GPG3 gel, and the same results as those for GPG1 gel were obtained.

[0080] Test Example 5. Rheology during Regelation (without Cells) GPG1 was dissolved in a 10 w / v% sucrose solution to a final concentration of 0.8 w / v%, and the resulting GPG1 solution was allowed to stand at 45°C for 1 day for initial gelation. The gel was converted into a sol by shaking with a Vortex mixer (Scientific Industries) for 5 seconds. An equal volume of 10 w / v% sucrose solution was added to the solution to obtain a sol with a peptide concentration of 0.4 w / v%. The gelation of these sols by heating them again is hereinafter referred to as "regelation." The rheology measurement method was the same as in Test Example 3. Immediately after removal from a 45°C incubator, the GPG1 sol (peptide concentration 0.4 w / v%) was placed in a rheometer MCR302 adjusted to 37°C, and dynamic viscoelasticity measurements were performed as in Test Example 4.

[0081] The results are shown in Figure 4. In the time-dispersion measurements shown in Figure 4A, G' consistently exceeded G" from the start of the measurement to the end. At the first point (after 30 seconds), G' was 233 Pa and G" was 28 Pa. G' and G" continued to decrease in a similar manner throughout the measurement, reaching 105 Pa and 13 Pa at the end of the measurement. In the strain-dispersion measurements shown in Figure 4B, G' and G" remained in a linear range, with approximately constant values, up to 17% strain. With further strain, G' increased and then decreased sharply beyond 41% strain. G" remained in a linear range up to 41% strain, after which it gradually decreased. No interchange of dominance between G' and G" was observed. In the frequency-dispersion measurements shown in Figure 4C, G' exceeded G" elastically throughout the entire measured frequency range from 0.1 to 100 rad / s. A gradual increase in G' and G" from the low frequency region to the high frequency region was observed, and G' was 10 1 ~10 2 Scale, G" is 10 0 ~10 1 The transition occurred on the scale, with tanδ (=G" / G') ranging from 0.116 to 0.295.

[0082] The same test was also performed on GPG3 gel, and the same results as those for GPG1 gel were obtained.

[0083] Test Example 6. Rheology during regelation (with cells) GPG1 was dissolved in a 10 w / v% aqueous sucrose solution to a final concentration of 0.8 w / v%, and the resulting GPG1 solution was allowed to stand at 45°C for one day to cause initial gelation. Each gel was converted into a sol by shaking it for 5 seconds using a vortex mixer (Scientific Industries). An equal volume of a cell suspension prepared by suspending Panc-1 cells in a 10 w / v% aqueous sucrose solution was added to the gel, and the resulting mixture was mixed at a peptide concentration of 0.4 w / v% and a cell concentration of 2.0 x 10 6 The sol was prepared at a concentration of 100 cells / mL. Measurement was carried out in the same manner as in Test Example 5 using the sol.

[0084] The results are shown in Figure 5. In the time-dispersion measurements shown in Figure 5A, G' consistently exceeded G" from the start of the measurement to the end. At the first point (after 30 seconds), G' was 10,578 Pa and G" was 2,913 Pa. Immediately after the start of the measurement, G' reached equilibrium and continued to increase slowly. G" showed a similar behavior, reaching 23,487 Pa and 4,742 Pa by the end of the measurement. In the strain-dispersion measurements shown in Figure 5B, G' and G" remained in a linear range, maintaining roughly constant values, up to approximately 2.84% strain. With further strain, G' decreased rapidly and G" decreased slowly, resulting in a switch in dominance between G' and G" at around 41% strain. In the frequency-dispersion measurements shown in Figure 5C, G' exceeded G" elastically over the entire measured frequency range of 0.1–100 rad / s. A gradual increase in G' and G" from the low frequency region to the high frequency region was observed, and G' was 10 4 On the scale, G" is 10 3 The transition occurred on the scale, with tanδ (=G" / G') ranging from 0.155 to 0.326.

[0085] When combined with the results of Test Example 5 without cells, it was found that suspending cells in the GPG sol / gel significantly improved the elastic modulus of the gel.

[0086] The same test was also performed on GPG3 gel, and the same results were obtained as for GPG1 gel. However, the improvement in gel elasticity due to cell suspension was not observed in the case of collagen gel.

[0087] Test Example 7. Three-dimensional cell culture using the thixotropic properties of the gel. Cell suspension sol obtained in the same manner as in Test Example 6 was dropped into a 96-well plate (Corning, Inc., USA) in 50 μL portions and allowed to stand in a 37°C incubator for 10 minutes to gel, creating a cell layer. 100 μL of cell culture medium (DMEM) was added dropwise from above, and the plates were cultured in a 37°C incubator with a 5% CO2 concentration for the appropriate time period. The cell layer was created on day 0. On days 1, 3, and 5, 50 μL of the supernatant cell culture medium was removed and replaced with 50 μL of fresh cell culture medium. On days 0, 2, 4, and 7, the gel was solized by pipetting with a micropipette and dispersed in PBS.

[0088] Cells in the gel and cells dispersed in PBS after solization were observed under a phase-contrast microscope on days 0, 2, 4, and 7. The images are shown in Figure 6. It was found that spheroids were formed by culturing using GPG gel. Furthermore, the thixotropy of GPG gel allowed for easy cell recovery and allowed for recovery without disrupting the spheroids.

[0089] Spheroids were also formed when three-dimensional culture was performed using collagen gel, but they could not be recovered by pipetting. They could be recovered after enzymatic treatment with collagenase.

[0090] Test Example 8: Two-dimensional cell culture NIH / 3T3 cells were cultured two-dimensionally on GPG1 gel at 37°C. After culturing, the cells were left at room temperature (approximately 15°C) for approximately 10 minutes, and the cells began to peel off in a sheet form. Because GPG is temperature responsive, it becomes hydrophilic at room temperature, which is thought to be why the cells peeled off. From the above, it was found that cell sheets can be easily recovered by two-dimensionally culturing cells on GPG gel.

[0091] Test Example 9. Three-dimensional cell culture using the thixotropy of gels (myoblast C2C12 cells) GPG3 was dissolved in 10 w / v% sucrose to a final concentration of 0.8 w / v%, and the resulting GPG3 solution was allowed to stand at 45°C for 2 days to initially gel. The gel was converted into a sol by shaking with a vortex mixer for 5 seconds. 50 μL of the sol was dropped into a cell culture vessel using a micropipette, and then 4 × 10 mouse-derived myoblast cell line C2C12 (RIKEN CELL BANK) cells were cultured. 6 50 μL of a suspension of 100 cells / mL dispersed in 10 w / v% sucrose was mixed and regelled. This resulted in a final GPG3 concentration of 0.4 w / v% and a cell concentration of 2 × 10 6 GPG3 gels were prepared at 0.1 cells / mL. One mL of DMEM medium containing 10% (v / v) fetal bovine serum (FBS) was layered on top, and culture was initiated at 37°C and 5% CO2. 300 μL of medium was replaced daily, and on day 3, the medium was switched to differentiation-inducing medium (DMEM medium containing 2% (v / v) horse serum). After differentiation induction, the cells were cultured for 7 days. Cell counts were assessed 4 hours and 3 days after the start of culture using the Cell Counting Kit-8 (CCK-8, DOJINDO). The medium was removed with a micropipette, and the remaining gel was pipetted to form a sol and collected in a microtube. The cells were then pelleted at 1000 rpm in a tabletop centrifuge. 100 μL of the supernatant was collected in a 96-well plate as a background, and the remaining supernatant was then discarded. The precipitated cells were dispersed in 150 μL of DMEM medium, and 100 μL of this solution was transferred to a 96-well plate. 10 μL of CCK solution was added to each well containing the background and sample. After incubation in a 37°C incubator for 3 hours, the absorbance at 450 nm was measured using a microplate reader (EPOCH2; BioTek). The cell proliferation rate was estimated by assuming that the difference in absorbance between the sample and background was proportional to the number of viable cells (Table 1).

[0092]

[0093] Seven days after the start of differentiation induction, muscle-like cells were observed within the gel using a phase-contrast microscope (BZ-X700, KEYENCE) (Fig. 7). Furthermore, the entire gel was immunostained for the skeletal muscle-specific expression marker α-actinin (Fig. 8), suggesting that myoblasts can be cultured within GPG3 gel and differentiated into skeletal muscle cells.

[0094] The immunostaining procedure was as follows. The medium was aspirated, and the gel was washed twice with 300 μL of phosphate-buffered saline (PBS). 400 μL of 4% paraformaldehyde in phosphate buffer was added, and the gel was incubated at 4°C for 30 minutes, followed by two washes with 300 μL of PBS. Polyoxyethylene (10) octylphenyl ether was diluted with PBS to a 0.5% solution, which was used as Triton X-100 solution. 400 μL of this solution was added to the gel, incubated at 4°C for 25 minutes, and then washed twice with 300 μL of PBS. Bovine serum-derived Cohn fraction V was diluted with PBS to a 1.0% solution, which was used as BSA solution. 400 μL of the BSA solution was added to the gel, incubated at room temperature for 1 hour, and then washed twice with 300 μL of PBS. 400 μL of Sarcomeric alpha actinin monoclonal antibody (EA-53, Thermo Fisher Scientific) diluted 200-fold with BSA solution was added as the primary antibody, and the plate was left to stand at 4°C for 16 hours. After washing twice with 300 μL of PBS, Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 1000 (AgF) was added to the BSA solution at a concentration of 0.1%. TM A secondary antibody, Plus 488 (Thermo Fisher Scientific), was added, and 400 μL of this was added to the gel. The gel was left to stand at room temperature for 1 hour in the dark, and then washed twice with 300 μL of PBS. NucBlue was also added to 2 mL of PBS. TM Fixed Cell ReadyProbes TM Four drops of the nuclear staining reagent (Thermo Fisher Scientific) were added to prepare a nuclear staining solution. 400 μL of this nuclear staining solution was added to the gel and left to stand at room temperature for 5 minutes in the dark. After washing once with 300 μL of PBS, the gel was observed under a fluorescence microscope (BZ-X700, KEYENCE).

Claims

1. (SEQ ID NO:1:X 1 GGX 2 G) n A G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 wherein m is the same or different and represents any amino acid, and m is an integer of 5 or greater.) A thixotropic gel or gel-forming sol composition containing a polypeptide comprising an elastin-like block peptide sequence comprising the elastin-like block peptide sequence, or a cell culture or proliferation substrate comprising a dried product of the composition.

2. The cell culture or proliferation substrate according to claim 1, wherein the sequence structure of the elastin-like block peptide sequence is, from the N-terminus, G sequence block-P sequence block-G sequence block, G sequence block-P sequence block-P sequence block-G sequence block, P sequence block-G sequence block, or G sequence block-P sequence block.

3. 2. The cell culture or growth substrate of claim 1 for use in three-dimensional culture or growth of cells.

4. 2. The cell culture or growth substrate of claim 1 for use in two-dimensional culture or growth of cells.

5. The cell culture or proliferation substrate of any one of claims 1 to 4, wherein the composition retains cells.

6. The cell culture or proliferation substrate according to any one of claims 1 to 4, wherein the concentration of the polypeptide in the composition is 0.2 w / v % or more.

7. The cell culture or proliferation substrate according to any one of claims 1 to 4, wherein the concentration of said polypeptide in said composition is 0.2 to 3 w / v %.

8. The cell culture or proliferation substrate according to any one of claims 1 to 4, for use after converting the gel-like composition into a sol and / or for use after converting the sol-like composition into a gel.

9. A cell culture or proliferation substrate according to any one of claims 1 to 4, for use in which the gel-like composition is converted into a sol by applying mechanical strain.

10. A method for culturing or growing cells, comprising culturing or growing cells on the cell culture or growth substrate according to any one of claims 1 to 4.

11. A cell population obtainable by the cell culture or proliferation method according to claim 10.