WO2015194494A1 - 脳損傷治療用細胞構造体、その製造方法、及び脳損傷治療剤 - Google Patents
脳損傷治療用細胞構造体、その製造方法、及び脳損傷治療剤 Download PDFInfo
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Definitions
- the present invention relates to a cell structure for treating brain injury, a method for producing the same, and a therapeutic agent for brain injury. Specifically, the present invention relates to a cell structure for treating brain damage in which a biocompatible polymer block is disposed in a gap between cells, a method for producing the same, and a therapeutic agent for brain damage.
- Regenerative medicine is being put to practical use to regenerate living tissues and organs that have fallen into dysfunction or dysfunction.
- Regenerative medicine is a new medicine that regenerates the same form and function as the original tissue using the three factors of cells, scaffolding, and growth factors from living tissue that can not be recovered only by the natural healing ability of the living body.
- Technology In recent years, treatment using cells is gradually being realized. For example, cultured epidermis using autologous cells, cartilage treatment using autologous chondrocytes, bone regeneration treatment using mesenchymal stem cells, cardiomyocyte sheet treatment using myoblasts, corneal regeneration treatment using corneal epithelial sheets, and Examples include nerve regeneration treatment.
- Patent Document 1 describes a cell structure including a polymer block having biocompatibility and cells, and a plurality of the polymer blocks arranged in the gaps between the plurality of cells.
- nutrients can be delivered from the outside to the inside of the cell structure.
- the cell structure has a sufficient thickness, and the cells are uniformly present in the structure.
- high cell survival activity has been demonstrated using a polymer block made of recombinant gelatin or a natural gelatin material.
- Patent Document 2 discloses a cell structure for cell transplantation, which includes a polymer block having biocompatibility and at least one type of cell, and a plurality of the polymer blocks are arranged in a gap between the plurality of cells. The body is listed.
- angiogenesis is evaluated using a cell structure for cell transplantation.
- Patent Document 3 describes a composition containing a cell physiologically active substance and cells, and describes that combination therapy with hepatocyte growth factor (HGF) and vascular endothelial cells is effective for regeneration of brain tissue.
- HGF hepatocyte growth factor
- Patent Document 4 describes an angiogenesis-inducing agent containing fibrin, a biodegradable polymer and cells.
- Patent Document 5 treats central nervous diseases such as stroke using a cell-containing composition in which bone marrow stromal cells into which Notch gene and / or Notch signaling-related gene is introduced are adhered to a biocompatible polymer. It is described to do.
- glutaraldehyde is used for polymer crosslinking.
- glutaraldehyde is used for polymer crosslinking.
- Patent Document 3 describes a combination therapy in which HGF and vascular endothelial cells are administered to brain tissue.
- improvement in motor function after cerebral infarction has not been evaluated, and a sufficient therapeutic effect on brain injury has been described. It is unclear whether it will be demonstrated.
- the angiogenesis-inducing agent described in Patent Document 4 requires the use of fibrin, and improvement in motor function after cerebral infarction has not been evaluated, and a sufficient therapeutic effect is exerted on brain damage. Whether or not it is unknown.
- the composition described in Patent Document 5 is obtained by culturing cells obtained by introducing Notch gene and / or Notch signaling-related gene into bone marrow stromal cells or medullary stromal cells under special conditions. The obtained cells are cells having specific properties.
- the present invention relates to a cell structure for treatment of brain injury, which does not contain glutaraldehyde, suppresses necrosis of transplanted cells (ie, has excellent cell survival rate), and can exhibit a sufficient therapeutic effect on brain damage, It was an object to provide a production method and a therapeutic agent for brain injury.
- the present inventors have used a biocompatible polymer block and at least one kind of cell, and a plurality of bioaffinity in a gap between a plurality of cells.
- the tap density is 10 mg / cm 3 or more and 500 mg / cm 3 or less, or the square root of the cross-sectional area in the two-dimensional cross-sectional image ⁇ perimeter
- a biocompatible polymer block having a value of 0.01 or more and 0.13 or less it is possible to provide a cell structure in which necrosis of transplanted cells is suppressed (that is, excellent in cell viability). I found it.
- the present inventors have found that the motor function of a rat can be improved by administering the above cell structure to a rat with cerebral infarction. The present invention has been completed based on these findings.
- a cell structure for treating brain damage comprising a biocompatible polymer block and at least one type of cell, wherein a plurality of the biocompatible polymer blocks are disposed in the gaps between the plurality of cells.
- the tap density of the biocompatible polymer block is 10 mg / cm 3 or more and 500 mg / cm 3 or less, or the square root of the cross-sectional area in the two-dimensional cross-sectional image of the biocompatible polymer block ⁇ surrounding
- a cell structure for treating brain damage wherein the length value is 0.01 or more and 0.13 or less.
- the biocompatible polymer block is composed of a recombinant peptide.
- the recombinant peptide is A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1; A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or 80% or more of the amino acid sequence described in SEQ ID NO: 1 A peptide having an amino acid sequence having the following sequence identity and having biocompatibility;
- the biocompatible polymer block is The biocompatible polymer solution is frozen by a freezing process in which the internal maximum liquid temperature, which is the liquid temperature of the highest liquid temperature in the solution, is 3 ° C lower than the solvent melting point in an unfrozen state.
- the biocompatible polymer block is The biocompatible polymer solution is frozen by a freezing process in which the internal maximum liquid temperature, which is the liquid temperature of the highest liquid temperature in the solution, is 3 ° C lower than the solvent melting point in an unfrozen state.
- (16) A cell structure for treating brain damage, which is obtained by fusing a plurality of cell structures for treating brain damage according to any one of (1) to (15).
- (17) A high biocompatibility in which the tap density is 10 mg / cm 3 or more and 500 mg / cm 3 or less, or the square root of the cross-sectional area in the two-dimensional cross-sectional image ⁇ perimeter length value is 0.01 or more and 0.13 or less
- a therapeutic agent for brain injury comprising the cell structure for treating brain injury according to any one of (1) to (16).
- a method for treating brain injury comprising a step of administering the cell structure for treating brain damage according to any one of (1) to (16) to a patient having brain injury.
- 20 Use of the cell structure for treating brain injury according to any one of (1) to (16) for the manufacture of a therapeutic agent for brain injury.
- the cell structure for brain injury treatment and the agent for treating brain injury according to the present invention can be manufactured without using glutaraldehyde, and thus is safe for the human body.
- the cell structure for brain injury treatment and the agent for treating brain injury according to the present invention suppresses necrosis of transplanted cells (that is, has excellent cell survival rate), and can effectively treat brain damage such as cerebral infarction. .
- FIG. 1 shows the block difference in the invitro ATP assay.
- FIG. 2 shows the difference in the survival state of cells in the hMSC mosaic cell mass using the block used in the present invention or the comparison block (after 2 weeks).
- FIG. 3 shows the survival state of transplanted cells between the block groups used in the present invention. The difference in survival and block size in hMSC mosaic cell mass is shown.
- FIG. 4 shows angiogenesis in hMSC mosaic cell mass 2 weeks after transplantation.
- FIG. 5 shows angiogenesis in hMSC + hECFC mosaic cell clusters 2 weeks after transplantation.
- FIG. 6 shows the space occupancy of each pore size in the porous body.
- FIG. 7 shows an HE cross-sectional image, pore shape, and internal maximum liquid temperature of the CBE3 porous body.
- FIG. 8 shows the time variation of the internal maximum liquid temperature at a shelf temperature of ⁇ 40 ° C. (glass plate 2.2 mm).
- FIG. 9 shows a liquid temperature profile during the production of the CBE3 porous body.
- FIG. 10 shows improvement in motor function by administration of GFP-expressing rat MSC mosaic cell cluster to cerebral infarction rats.
- FIG. 11 shows improvement in motor function by administration of rat bone marrow cell mosaic cell mass to cerebral infarction rats and administration of hMSC mosaic cell mass to cerebral infarction-induced nude rats.
- FIG. 12 shows the results of examining the time required for formation of the mosaic cell mass.
- the present invention relates to a brain injury treatment cell comprising a biocompatible polymer block and at least one kind of cell, wherein a plurality of the biocompatible polymer blocks are arranged in a gap between the plurality of cells.
- the present invention relates to a cell structure for treating brain injury, the perimeter of which is 0.01 or more and 0.13 or less, a method for producing the same, and a therapeutic agent for brain injury comprising the cell structure for treating brain damage.
- the cell structure in the present invention may be referred to as a mosaic cell mass (a mosaic cell mass) in the present specification.
- Bioaffinity polymer block (1-1) Bioaffinity polymer Bioaffinity refers to significant adverse reactions such as long-term and chronic inflammatory reactions when in contact with the living body. Does not provoke.
- the biocompatible polymer used in the present invention is not particularly limited as to whether or not it is degraded in vivo as long as it has affinity for the living body, but is preferably a biodegradable polymer.
- Specific examples of non-biodegradable materials include polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC (2-methacryloyloxyethyl phosphorylcholine). It is done.
- biodegradable materials include polypeptides such as recombinant peptides (for example, gelatin described below), polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer (PLGA), hyaluronic acid, glycosamino Examples include glycans, proteoglycans, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, and chitosan. Among the above, a recombinant peptide is particularly preferable. These biocompatible polymers may be devised to enhance cell adhesion.
- recombinant peptides for example, gelatin described below
- polylactic acid polyglycolic acid
- PLGA lactic acid / glycolic acid copolymer
- hyaluronic acid glycosamino Examples include glycans, proteoglycans, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, and chitosan
- Cell adhesion substrate fibronectin, vitronectin, laminin
- cell adhesion sequence expressed by one letter code of amino acid, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence
- Coating with peptide “Amination of substrate surface, cationization”, or “Plasma treatment of substrate surface, hydrophilic treatment by corona discharge”, etc. You can use the method.
- the type of polypeptide containing the recombinant peptide is not particularly limited as long as it has biocompatibility. And most preferred are gelatin, collagen and atelocollagen.
- the gelatin for use in the present invention is preferably natural gelatin or recombinant gelatin, and more preferably recombinant gelatin.
- natural gelatin means gelatin made from naturally derived collagen. Recombinant gelatin will be described later in this specification.
- the hydrophilicity value “1 / IOB” value of the biocompatible polymer used in the present invention is preferably from 0 to 1.0. More preferably, it is 0 to 0.6, and still more preferably 0 to 0.4.
- IOB is an index of hydrophilicity / hydrophobicity based on an organic conceptual diagram representing the polarity / nonpolarity of an organic compound proposed by Satoshi Fujita, and details thereof are described in, for example, “Pharmaceutical Bulletin”, vol.2, 2, pp .163-173 (1954), “Area of Chemistry” vol.11, 10, pp.719-725 (1957), “Fragrance Journal”, vol.50, pp.79-82 (1981), etc. Yes.
- methane (CH 4 ) is the source of all organic compounds, and all the other compounds are all methane derivatives, with certain numbers set for their carbon number, substituents, transformations, rings, etc. Then, the score is added to obtain an organic value (OV) and an inorganic value (IV), and these values are plotted on a diagram with the organic value on the X axis and the inorganic value on the Y axis. It is going.
- the IOB in the organic conceptual diagram refers to the ratio of the inorganic value (IV) to the organic value (OV) in the organic conceptual diagram, that is, “inorganic value (IV) / organic value (OV)”.
- hydrophilicity / hydrophobicity is represented by a “1 / IOB” value obtained by taking the reciprocal of IOB. The smaller the “1 / IOB” value (closer to 0), the more hydrophilic it is.
- the hydrophilicity is high and the water absorption is high. It is presumed that this contributes to the stabilization and survival of cells in the cell structure (mosaic cell mass).
- the biocompatible polymer used in the present invention is a polypeptide
- the hydrophilicity / hydrophobicity index represented by Grand ⁇ average of hydropathicity (GRAVY) value 0.3 or less, preferably minus 9.0 or more, More preferably, it is 0.0 or less and minus 7.0 or more.
- Grand average of hydropathicity (GRAVY) values are: Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins MR, Appel RD, Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). Pp.
- the GRAVY value of the biocompatible polymer used in the present invention in the above range, the hydrophilicity is high and the water absorption is high. It is presumed to contribute to the stabilization and survival of cells in the body (mosaic cell mass).
- the biocompatible polymer used in the present invention may be cross-linked or non-cross-linked, but is preferably cross-linked.
- a cross-linked biocompatible polymer By using a cross-linked biocompatible polymer, it is possible to obtain an effect of preventing instantaneous degradation when cultured in a medium or transplanted to a living body.
- Common crosslinking methods include thermal crosslinking, crosslinking with aldehydes (eg, formaldehyde, glutaraldehyde, etc.), crosslinking with condensing agents (carbodiimide, cyanamide, etc.), enzyme crosslinking, photocrosslinking, UV crosslinking, hydrophobic interaction, Hydrogen bonds, ionic interactions, etc. are known.
- the biocompatible polymer block in the present invention is preferably a biocompatible polymer block that does not contain glutaraldehyde, and more preferably a biocompatible polymer block that does not contain an aldehyde or a condensing agent.
- the crosslinking method used in the present invention is more preferably thermal crosslinking, ultraviolet crosslinking, or enzyme crosslinking, and particularly preferably thermal crosslinking.
- the enzyme When performing cross-linking with an enzyme, the enzyme is not particularly limited as long as it has a cross-linking action between polymer materials.
- trans-glutaminase and laccase most preferably trans-glutaminase can be used for cross-linking.
- a specific example of a protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it has a lysine residue and a glutamine residue.
- the transglutaminase may be derived from a mammal or may be derived from a microorganism. Specifically, transglutaminase derived from a mammal that has been marketed as an Ajinomoto Co., Ltd.
- Human-derived blood coagulation factors Factor XIIIa, Haematologic Technologies, Inc.
- Factor XIIIa Haematologic Technologies, Inc.
- guinea pig liver-derived transglutaminase goat-derived transglutaminase
- rabbit-derived transglutaminase from Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign Bio International, etc. Etc.
- the reaction temperature at the time of performing crosslinking is not particularly limited as long as crosslinking is possible, but is preferably ⁇ 100 ° C. to 500 ° C., more preferably 0 ° C. to 300 ° C., and still more preferably. It is 50 ° C to 300 ° C, more preferably 100 ° C to 250 ° C, and further preferably 120 ° C to 200 ° C.
- the recombinant gelatin referred to in the present invention means a polypeptide or protein-like substance having an amino acid sequence similar to gelatin produced by a gene recombination technique.
- the recombinant gelatin that can be used in the present invention preferably has a repeating sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents an amino acid).
- Gly-XY may be the same or different.
- two or more cell adhesion signals are contained in one molecule.
- recombinant gelatin used in the present invention recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used.
- EP1014176, US Pat. No. 6,992,172, International Publication WO2004 / 85473, International Publication WO2008 / 103041, and the like can be used, but are not limited thereto.
- a preferable example of the recombinant gelatin used in the present invention is the recombinant gelatin of the following embodiment.
- Recombinant gelatin has excellent biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived, so there is no concern about bovine spongiform encephalopathy (BSE) and excellent non-infectivity. Recombinant gelatin is more uniform than natural ceratin and its sequence is determined, so that strength and degradability can be precisely designed with less blur due to cross-linking and the like.
- BSE bovine spongiform encephalopathy
- the molecular weight of the recombinant gelatin is not particularly limited, but is preferably 2 kDa or more and 100 kDa or less, more preferably 2.5 kDa or more and 95 kDa or less, further preferably 5 kDa or more and 90 kDa or less, and most preferably 10 kDa or more and 90 kDa or less. is there.
- Recombinant gelatin preferably has a repeating sequence represented by Gly-XY characteristic of collagen.
- the plurality of Gly-XY may be the same or different.
- Gly-XY Gly represents glycine
- X and Y represent any amino acid (preferably any amino acid other than glycine).
- the sequence represented by Gly-XY, which is characteristic of collagen, is a very specific partial structure in the amino acid composition and sequence of gelatin / collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats.
- Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation.
- the amino acids represented by X and Y are rich in imino acids (proline, oxyproline), and preferably account for 10% to 45% of the total.
- 80% or more, more preferably 95% or more, and most preferably 99% or more of the amino acid sequence of the recombinant gelatin is a Gly-XY repeating structure.
- polar amino acids are charged and uncharged at 1: 1.
- the polar amino acid specifically refers to cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine and arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. Refers to threonine and tyrosine.
- the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids.
- the proportion of uncharged amino acids in the polar amino acid is preferably 5% or more and less than 20%, preferably less than 10%. Furthermore, it is preferable that any one amino acid, preferably two or more amino acids among serine, threonine, asparagine, tyrosine and cysteine are not included in the sequence.
- the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology”, Vol. 9, No. 7 (1990), page 527, published by Nagai Publishing Co., Ltd.).
- the recombinant gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule.
- Specific sequences include RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LGITIPG sequences, RNIAEIIKDI sequences, which are expressed in one-letter amino acid notation in that many types of cells adhere.
- IKVAV sequences IKVAV sequences, LRE sequences, DGEA sequences, and HAV sequences are preferred. More preferred are RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence and HAV sequence, and particularly preferred is RGD sequence. Of the RGD sequences, an ERGD sequence is preferred.
- the amount of cell substrate produced can be improved. For example, in the case of cartilage differentiation using mesenchymal stem cells as cells, production of glycosaminoglycan (GAG) can be improved.
- GAG glycosaminoglycan
- the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.
- the content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 per protein molecule from the viewpoint of cell adhesion / proliferation. Most preferably, it is 12.
- the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%.
- each stretch of 350 amino acids contains at least one RGD motif.
- the ratio of RGD motif to the total number of amino acids is more preferably at least 0.6%, more preferably at least 0.8%, more preferably at least 1.0%, more preferably at least 1.2%. And most preferably at least 1.5%.
- the number of RGD motifs in the recombinant peptide is preferably at least 4, more preferably 6, more preferably 8, more preferably 12 or more and 16 or less per 250 amino acids.
- a ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature.
- the recombinant gelatin used in the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains one RGD sequence. As a further aspect of the recombinant gelatin used in the present invention, it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.
- Recombinant gelatin may be partially hydrolyzed.
- the recombinant gelatin used in the present invention is represented by the formula: A-[(Gly-XY) n ] m -B.
- n Xs independently represents any of amino acids
- n Ys independently represents any of amino acids.
- m is preferably 2 to 10, and preferably 3 to 5.
- n is preferably 3 to 100, more preferably 15 to 70, and most preferably 50 to 65.
- A represents any amino acid or amino acid sequence
- B represents any amino acid or amino acid sequence
- n Xs each independently represent any amino acid
- n Ys each independently represent any amino acid. Show.
- the recombinant gelatin used in the present invention has the formula: Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly (wherein 63 X independently represent any of the amino acids). 63 Y's each independently represent any of the amino acids, wherein 63 Gly-XY may be the same or different.
- the naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, or type V collagen. More preferred is type I, type II, or type III collagen.
- the collagen origin is preferably human, bovine, porcine, mouse or rat, more preferably human.
- the isoelectric point of the recombinant gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.
- the recombinant gelatin is not deaminated.
- the recombinant gelatin has no telopeptide.
- the recombinant gelatin is a substantially pure polypeptide prepared with a nucleic acid encoding an amino acid sequence.
- a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (2) A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having biocompatibility; or (3) described in SEQ ID NO: 1 A peptide consisting of an amino acid sequence having a sequence identity of 80% or more (more preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more) with the amino acid sequence and having biocompatibility;
- amino acid sequence in which one or several amino acids are deleted, substituted or added is preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5. Means, particularly preferably 1 to 3.
- Recombinant gelatin used in the present invention can be produced by genetic recombination techniques known to those skilled in the art. For example, EP 1014176A2, US Pat. It can be produced according to the method described. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is introduced into an appropriate host to produce a transformant. . Recombinant gelatin is produced by culturing the obtained transformant in an appropriate medium. Therefore, the recombinant gelatin used in the present invention can be prepared by recovering the recombinant gelatin produced from the culture. .
- Bioaffinity polymer block In the present invention, a block (lumb) made of the above-described bioaffinity polymer is used.
- the shape of the biocompatible polymer block in the present invention is not particularly limited. For example, amorphous, spherical, particulate, powdery, porous, fibrous, spindle-shaped, flat and sheet-like, preferably amorphous, spherical, particulate, powdery and porous More preferably, it is indefinite.
- An indeterminate shape indicates that the surface shape is not uniform, for example, an object having irregularities such as rocks.
- the tap density of the biocompatible polymer block in the present invention is 10 mg / cm 3 or more and 500 mg / cm 3 or less, preferably 20 mg / cm 3 or more and 400 mg / cm 3 or less, more preferably 40 mg / cm 3 or more and 220 mg. / Cm 3 or less, more preferably 50 mg / cm 3 or more and 150 mg / cm 3 or less.
- the tap density is a value that indicates how many blocks can be densely packed in a certain volume. It can be seen that the smaller the value, the more densely packed, that is, the more complicated the block structure.
- the tap density of the biocompatible polymer block represents the complexity of the surface structure of the biocompatible polymer block and the amount of voids formed when the biocompatible polymer block is collected as an aggregate. It is conceivable that. The smaller the tap density, the more voids between the polymer blocks and the larger the cell engraftment area.
- the tap density as used in this specification can be measured as follows.
- a container hereinafter referred to as a cap
- a funnel is attached to the cap and the block is poured from the funnel so that the block accumulates in the cap.
- tap the cap part 200 times on a hard place such as a desk, remove the funnel and clean with a spatula.
- the mass is measured with the cap fully filled.
- “Square root of cross-sectional area ⁇ perimeter” of the biocompatible polymer block in the present invention is 0.01 or more and 0.13 or less, preferably 0.02 or more and 0.12 or less, more preferably 0.03. It is not less than 0.115, more preferably not less than 0.05 and not more than 0.09.
- the “square root of cross-sectional area ⁇ perimeter” of the biocompatible polymer block means the complexity of the surface structure of the biocompatible polymer block as well as the tap density, and when the polymer blocks are collected as an aggregate. It is thought that it represents the amount of voids formed.
- there can be a moderately biocompatible polymer block between cells and in the case of a cell structure for cell transplantation, nutrients can be delivered to the inside of the structure, It is considered that it is preferable to be within the above range.
- the “square root of the area ⁇ perimeter” in the two-dimensional cross-sectional image of the biocompatible polymer block can be obtained by preparing a cross-sectional specimen of the biocompatible polymer block and confirming the cross-sectional structure.
- the cross-sectional structure of the biocompatible polymer block is prepared as a sliced specimen (for example, a HE-stained specimen).
- the biocompatible polymer block may be used, or the cross-sectional structure may be observed as a cell structure including the biocompatible polymer block and cells.
- its cross-sectional area and perimeter are obtained, and then “square root of cross-sectional area ⁇ perimeter” is calculated. It can be measured over a plurality of 10 or more locations, and the average value of them can be obtained as “square root of sectional area ⁇ perimeter”.
- the size of one biocompatible polymer block in the present invention is not particularly limited, but is preferably 1 ⁇ m or more and 700 ⁇ m or less, more preferably 10 ⁇ m or more and 700 ⁇ m or less, further preferably 10 ⁇ m or more and 300 ⁇ m or less, Preferably they are 20 micrometers or more and 200 micrometers or less, More preferably, they are 20 micrometers or more and 150 micrometers or less, Especially preferably, they are 25 micrometers or more and 106 micrometers or less, and it is also preferable that they are 50 micrometers or more and 120 micrometers or less.
- the size of one biocompatible polymer block does not mean that the average value of the sizes of a plurality of biocompatible polymer blocks is in the above range. It means the size of each biocompatible polymer block obtained by sieving the block.
- the size of one block can be defined by the size of the sieve used to separate the blocks.
- a block remaining on the sieve when the passed block is passed through a 106 ⁇ m sieve after passing through a 180 ⁇ m sieve can be a block having a size of 106 to 180 ⁇ m.
- the block remaining on the sieve when the block passed through the sieve of 106 ⁇ m and the passed block is passed through the sieve of 53 ⁇ m can be a block having a size of 53 to 106 ⁇ m.
- a block remaining on the sieve when the passed block is passed through a sieve of 53 ⁇ m and passed through a sieve of 25 ⁇ m can be made a block having a size of 25 to 53 ⁇ m.
- Method for producing biocompatible polymer block is particularly limited as long as a biocompatible polymer block satisfying the conditions described in (1-4) above is obtained.
- a porous body of a biocompatible polymer can be pulverized using a pulverizer (such as a new power mill) to form a granule, whereby the conditions described in the above (1-4) A biocompatible polymer block that satisfies the requirements can be obtained.
- the “porous body” in the present invention is preferably a material having a plurality of “pores of 10 ⁇ m or more and 500 ⁇ m or less” inside the main body when prepared as a 1 mm square material, and voids in the main body.
- a material having an occupied volume of 50% or more can be used.
- the internal vacancies may be in communication with each other, or some or all of the vacancies may be open on the material surface.
- the liquid temperature at the highest liquid temperature in the solution is 3 ° C. lower than the solvent melting point in an unfrozen state (“solvent By including a freezing step that has a melting point of ⁇ 3 ° C. ”) or less, the formed ice becomes spherical. Through this step, the ice is dried to obtain a porous body having spherical isotropic pores (spherical holes). Does not include the freezing step in which the liquid temperature at the highest temperature in the solution (internal maximum liquid temperature) is 3 ° C lower than the solvent melting point (“solvent melting point-3 ° C") or more in the unfrozen state. By freezing, the formed ice becomes a pillar / flat plate. When the ice is dried through this step, a porous body having columnar or plate-like pores (column / plate hole) that is long on one or two axes is obtained.
- the shape of the pores of the porous body is preferably a spherical hole rather than a column / plate hole, and the proportion of the spherical holes in the pores is 50% or more. More preferably.
- the internal maximum liquid temperature which is the liquid temperature of the highest liquid temperature in the solution, is 3 ° C. lower than the solvent melting point in an unfrozen state (“solvent melting point ⁇ 3 ° C.”).
- a porous body of a biocompatible polymer can be produced by a method comprising This is because, according to the above process, the ratio of the sphere holes in the holes can be 50% or more.
- pulverizes the porous body obtained at the said process b can be included.
- the internal maximum liquid temperature which is the liquid temperature of the highest liquid temperature in the solution, is 7 ° C. lower than the solvent melting in an unfrozen state (“solvent melting point ⁇ 7 ° C.”). It can be frozen by the following freezing process. This is because, according to this step, the proportion of the spherical holes in the holes can be 80% or more.
- the average pore size of the pores of the porous body can be obtained from observation of the cross-sectional structure of the porous body.
- the cross-sectional structure of the porous body is prepared as a sliced specimen (for example, a HE (hematoxylin / eosin) -stained specimen).
- the clear protrusions of the walls formed of the polymer are connected to the closest protrusions to clarify the holes.
- the area of each divided hole thus obtained is measured, and then the circle diameter when the area is converted into a circle is calculated.
- the obtained circular diameter can be used as the pore size, and an average value of 20 or more can be used as the average pore size.
- the space occupation ratio of a certain pore size means the ratio of the volume of pores having a certain pore size in the porous body. Specifically, it can be obtained as a ratio by dividing the area occupied by holes of a certain hole size by the total area from the two-dimensional cross-sectional image. Moreover, as a cross-sectional image to be used, a cross-sectional image with an actual size of 1.5 mm can be used.
- the space occupation ratio occupied by the pore size of 20 ⁇ m to 200 ⁇ m of the porous body is preferably 83% or more and 100% or less, more preferably 85% or more and 100% or less, still more preferably 90% or more and 100% or less, and particularly preferably. Is 95% or more and 100% or less.
- the space occupation ratio occupied by the pore size of 30 ⁇ m to 150 ⁇ m of the porous body is preferably 60% or more and 100% or less, more preferably 70% or more and 100% or less, still more preferably 80% or more and 100% or less, and particularly preferably Is 90% or more and 100% or less.
- the space occupancy occupied by the pore size of 20 ⁇ m to 200 ⁇ m of the porous body and the space occupancy occupied by the pore size of 30 ⁇ m to 150 ⁇ m of the porous body are such that the pore size distribution in the porous body falls within a predetermined range.
- the structure of the polymer block after pulverization becomes complicated, and as a result, the tap density and “square root of the cross-sectional area ⁇ perimeter” are reduced.
- the major axis and the minor axis are obtained for each hole, and “major axis ⁇ minor axis” is calculated therefrom.
- “major axis ⁇ short axis” is 1 or more and 2 or less, it can be a spherical hole, and when it is 3 or more, it can be a column / plate hole.
- the bulk density ( ⁇ ) is calculated from the dry mass and the volume, and the true density ( ⁇ c) can be determined by the Hubbard-type specific gravity bottle method.
- the porosity of the polymer porous body in the present invention is preferably 81% or more and 99.99% or less, more preferably 95.01% or more and 99.9% or less.
- any cell can be used as long as it can treat brain damage, and the type thereof is not particularly limited. Further, one type of cell may be used, or a plurality of types of cells may be used in combination.
- the cells to be used are preferably animal cells, more preferably vertebrate cells, and particularly preferably human cells. Vertebrate-derived cells (particularly human-derived cells) may be any of universal cells, somatic stem cells, progenitor cells, or mature cells. For example, embryonic stem (ES) cells, reproductive stem (GS) cells, or induced pluripotent stem (iPS) cells can be used as the universal cells.
- ES embryonic stem
- GS reproductive stem
- iPS induced pluripotent stem
- somatic stem cells for example, mesenchymal stem cells (MSC), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow cells (bone marrow-derived cells), myocardial stem cells, adipose-derived stem cells, or neural stem cells can be used.
- MSC mesenchymal stem cells
- hematopoietic stem cells amniotic cells
- umbilical cord blood cells bone marrow cells (bone marrow-derived cells)
- myocardial stem cells adipose-derived stem cells
- neural stem cells for example, mesenchymal stem cells (MSC), hematopoietic stem cells, amniotic cells, umbilical cord blood cells, bone marrow cells (bone marrow-derived cells), myocardial stem cells, adipose-derived stem cells, or neural stem cells
- progenitor cells and mature cells for example, cells derived from nerves, brain, or bone marrow can
- vascular cells can also be used.
- the vascular cell means a cell related to angiogenesis, and is a cell constituting blood vessels and blood, and a progenitor cell and a somatic stem cell that can differentiate into the cell.
- the vascular cells include cells that do not naturally differentiate into cells that constitute blood vessels and blood such as ES cells, GS cells, or iPS cells, and mesenchymal stem cells (MSC). Not included.
- the vascular system cell is preferably a cell constituting a blood vessel.
- specific examples of cells constituting blood vessels include vascular endothelial cells and vascular smooth muscle cells.
- Vascular endothelial cells may be either venous endothelial cells or arterial endothelial cells.
- Vascular endothelial progenitor cells can be used as progenitor cells for vascular endothelial cells.
- Vascular endothelial cells and vascular endothelial progenitor cells are preferred.
- As cells constituting blood blood cells can be used, white blood cells such as lymphocytes and neutrophils, monocytes, and hematopoietic stem cells which are stem cells thereof can be used.
- the non-vascular cell means a cell other than the above-mentioned vascular cell.
- ES cells iPS cells, mesenchymal stem cells (MSC), or nerve cells can be used.
- MSC mesenchymal stem cells
- nerve cells preferably, MSC or iPS cells can be used. More preferably, it is MSC.
- a cell expressing a nervous system gene can be used, and preferably, an MSC expressing a nervous system gene can be used.
- Neural genes include Sox2, Nestin (Nestin), NeuroD1 (Neurogenic differentiation 1), GAD1 (GABA synthesis), GRIA1 (glutamate receptor 1), GRIA2 (glutamate receptor 2), CHRM1 (acetylcholine receptor 1) , GABRA1 (GABAA receptor ⁇ 1), GABBR1 (GABAB receptor 1), CHAT (acetylcholine synthesis), DDC (serotonin / DOPA synthesis), HTR1A (serotonin receptor 1A), HTR1B (serotonin receptor 1B), HTR2A (serotonin) Receptor 2A), 5-HTT (serotonin transporter), Ascl1 (neural stem cell neuron differentiation marker), Hes1 (neural stem cell astrocyte differentiation marker), and Olig2 (neural stem).
- the cells used in the present invention there is no particular limitation.
- the cells used in the present invention one or more of the above-mentioned genes of the nervous system, preferably 2 or more, more preferably 3 or more, further preferably 5 or more, more preferably 7 or more, particularly preferably Can be used cells expressing 10 or more types, most preferably 13 or more types (preferably MSCs).
- one or more genes selected from Sox2, Nestin, NeurD1, GAD1, GRIA1, GRIA2, GABRA1, GABBR1, DDC, HTR1B, HTR2A, 5-HTT, Hes1, preferably two or more, more preferably Cells expressing 3 or more, more preferably 5 or more, more preferably 7 or more, particularly preferably 10 or more, and most preferably all 13 types (preferably MSC) can be used.
- MSC preferably MSC that does not express the above gene or an MSC that expresses a nervous system gene other than the above gene can also be used.
- a method for measuring the expression state (presence / absence and level of expression) of a specific gene in a cell is known to those skilled in the art, and is commonly used such as RT-PCR (reverse transcription polymerase chain reaction) and Northern blotting. It can be measured by the method.
- a plurality of biocompatible polymer blocks are mosaic-shaped in a space between a plurality of cells using biocompatible polymer blocks and cells. It is possible to have a thickness suitable for cell transplantation by arranging in a dimension. Further, the biocompatible polymer block and the cells are arranged in a three-dimensional mosaic manner to form a cell structure in which the cells are uniformly present in the structure, and from outside to inside the cell structure. Nutritional delivery is possible.
- necrosis of the transplanted cells is suppressed and transplantation becomes possible.
- “suppression of necrosis” means that the degree of necrosis is low as compared with the case of transplanting only cells without using a cell structure.
- a plurality of biocompatible polymer blocks are arranged in a gap between a plurality of cells.
- the “gap between cells” It is not necessary that the space is closed by the cells to be closed, as long as it is sandwiched between the cells. Note that there is no need for a gap between all cells, and there may be a place where the cells are in contact with each other.
- the gap distance between the cells via the biocompatible polymer block that is, the gap distance when selecting a cell and a cell that is present at the shortest distance from the cell is not particularly limited.
- the size is preferably the size of the polymer block, and the preferred distance is also within the preferred size range of the biocompatible polymer block.
- the biocompatible polymer block is sandwiched between cells, but there is no need for cells between all the biocompatible polymer blocks, and the biocompatible polymer blocks are in contact with each other. There may be.
- the distance between the biocompatible polymer blocks via the cells that is, the distance when the biocompatible polymer block and the biocompatible polymer block existing at the shortest distance from the biocompatible polymer block are selected are particularly Although not limited, it is preferably the size of a cell mass when one to several cells used are collected, for example, 10 ⁇ m or more and 1000 ⁇ m or less, preferably 10 ⁇ m or more and 100 ⁇ m or less. More preferably, it is 10 ⁇ m or more and 50 ⁇ m or less.
- the expression “is uniformly present” such as “a cell structure in which cells are uniformly present in the structure” is used, but does not mean complete uniformity, It means that nutrition can be delivered from the outside to the inside of the cell structure, and the necrosis of the transplanted cells is prevented.
- the thickness or diameter of the cell structure for treating brain damage can be set to a desired thickness, but the lower limit is preferably 215 ⁇ m or more, more preferably 400 ⁇ m or more, and most preferably 730 ⁇ m or more. .
- the upper limit of the thickness or diameter is not particularly limited, but the general range for use is preferably 3 cm or less, more preferably 2 cm or less, and even more preferably 1 cm or less.
- the range of the thickness or diameter of the cell structure for treating brain damage is preferably 400 ⁇ m or more and 3 cm or less, more preferably 500 ⁇ m or more and 2 cm or less, and further preferably 720 ⁇ m or more and 1 cm or less.
- a region composed of a biocompatible polymer block and a region composed of cells are arranged in a mosaic pattern.
- the “thickness or diameter of the cell structure for treatment of brain injury” in this specification means the following.
- the ratio of cells to the biocompatible polymer block is not particularly limited, but the ratio of the biocompatible polymer block per cell is preferably 0.0000001 ⁇ g or more and 1 ⁇ g. Or less, more preferably 0.000001 ⁇ g to 0.1 ⁇ g, more preferably 0.00001 ⁇ g to 0.01 ⁇ g, and most preferably 0.00002 ⁇ g to 0.006 ⁇ g.
- the ratio of the cells to the biocompatible polymer block within the above range, the cells can be present more uniformly.
- the components in the biocompatible polymer block that are optionally present are cells.
- the component in the biocompatible polymer block is not particularly limited, and examples thereof include components contained in the medium described later.
- the cell structure for treatment of brain injury of the present invention may contain an angiogenic factor.
- the angiogenic factor include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and the like.
- bFGF basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- HGF hepatocyte growth factor
- the manufacturing method of the cell structure containing an angiogenic factor is not particularly limited, for example, it can be manufactured by using a biocompatible polymer block impregnated with an angiogenic factor. From the viewpoint of promoting angiogenesis, the cell structure for treatment of brain injury of the present invention preferably contains an angiogenic factor.
- the cell structure for treatment of brain injury of the present invention may contain non-vascular cells. Further, the cells constituting the cell structure may be only non-vascular cells. A cell structure containing only non-vascular cells as cells can form blood vessels at the site of transplantation after transplantation. In addition, when there are two or more types of cells constituting the cell structure and both non-vascular cells and vascular cells are included, more blood vessels than in the case where only non-vascular cells are formed. It can be formed and is preferable.
- the cell structure for treatment of brain injury according to the present invention is preferably one in which blood vessels are formed inside the cell structure.
- the cell structure for treatment of brain injury includes a cell structure containing two or more types of cells and including both non-vascular cells and vascular cells. Including those formed.
- the cell structure for treatment of brain damage of the present invention can be produced by mixing a biocompatible polymer block and at least one kind of cell. More specifically, the cell structure for treatment of brain injury according to the present invention can be produced by alternately arranging biocompatible polymer blocks and cells.
- the production method is not particularly limited, but is preferably a method of seeding cells after forming a biocompatible polymer block.
- the cell structure for treatment of brain injury of the present invention can be produced by incubating a mixture of a biocompatible polymer block and a cell-containing culture solution.
- cells and a biocompatible polymer block prepared in advance are arranged in a mosaic in a container and in a liquid held in the container.
- it is preferable to promote or control the formation of a mosaic array composed of cells and a biocompatible substrate by using natural aggregation, natural dropping, centrifugation, and stirring.
- a living body having a tap density of 10 mg / cm 3 or more and 500 mg / cm 3 or less, or a square root of a cross-sectional area divided by a peripheral length of 0.01 to 0.13 in a two-dimensional cross-sectional image.
- a method for producing a cell structure for treating brain injury according to the present invention comprising a step of mixing an affinity polymer block and cells and culturing for 10 hours or more. That is, the cell structure for treatment of brain injury of the present invention is preferably obtained by mixing a biocompatible polymer block and cells and culturing for 10 hours or more, and by culturing for 12 hours or more.
- the container used is preferably a container made of a low cell adhesion material or a cell non-adhesive material, and more preferably a container made of polystyrene, polypropylene, polyethylene, glass, polycarbonate, or polyethylene terephthalate.
- the shape of the bottom surface of the container is preferably a flat bottom type, a U shape, or a V shape.
- the cell structure (mosaic cell mass) obtained by the above method is, for example, (A) fusing separately prepared cell structures (mosaic cell masses), or (b) increasing the volume under differentiation medium or growth medium, A cell structure of a desired size can be produced by such a method.
- the fusion method and the volume increase method are not particularly limited.
- the cell structure in the step of incubating the mixture of the biocompatible polymer block and the cell-containing culture solution, can be increased in volume by exchanging the medium with a differentiation medium or a growth medium.
- a cell structure having a desired size is obtained by further adding the biocompatible polymer block. A cell structure in which cells are uniformly present in the body can be produced.
- a plurality of biocompatible polymer blocks and a plurality of cells When fusing separately prepared cell structures, for example, a plurality of biocompatible polymer blocks and a plurality of cells, and a part of a plurality of gaps formed by the plurality of cells Alternatively, a plurality of cell structures in which one or a plurality of the above-mentioned biocompatible polymer blocks are arranged can be fused together.
- a cell structure obtained by fusing a plurality of cell structures as described in (a) above can also be used as the cell structure for treating brain damage of the present invention.
- Biocompatible polymer block (type, size, etc.)”, “cell”, “gap between cells”, “obtained cell structure (size) in the method for producing a cell structure for brain injury treatment of the present invention Etc. ”and“ ratio of cells and biocompatible polymer block ”and the like are the same as described above in the present specification.
- the thickness or diameter of each cell structure before fusion is preferably 10 ⁇ m or more and 1 cm or less, more preferably 10 ⁇ m or more and 2000 ⁇ m or less, still more preferably 15 ⁇ m or more and 1500 ⁇ m or less, and most preferably 20 ⁇ m or more and 1300 ⁇ m or less.
- the thickness or diameter after fusion is preferably 400 ⁇ m or more and 3 cm or less, more preferably 500 ⁇ m or more and 2 cm or less, and further preferably 720 ⁇ m or more and 1 cm or less.
- biocompatible polymer block specifically, a plurality of first biocompatible polymer blocks
- a method of adding and incubating the second biocompatible polymer block can be mentioned.
- biocompatible polymer block (type, size, etc.)”, “cell”, “gap between cells”, “obtained cell structure (size, etc.)”, “cell and bioaffinity high Suitable ranges such as “ratio of molecular blocks” are the same as those described above in the present specification.
- the cell structures to be fused are preferably placed at a distance of 0 to 50 ⁇ m, more preferably 0 to 20 ⁇ m, and still more preferably 0 to 5 ⁇ m.
- the cells or the substrate produced by the cells by the growth and expansion of the cells will act as an adhesive and be joined. Becomes easy.
- the range of the thickness or diameter of the cell structure obtained as described above is preferably 400 ⁇ m or more and 3 cm or less, more preferably 500 ⁇ m or more and 2 cm or less, and further preferably 720 ⁇ m or more and 1 cm or less.
- the pace at which the second biocompatible polymer block is added depends on the growth rate of the cells used. It is preferable to select appropriately. Specifically, if the pace at which the second biocompatible polymer block is added is fast, the cells move to the outside of the cell structure, resulting in poor cell uniformity. Since a portion where the ratio is increased is formed and the uniformity of the cells is lowered, selection is made in consideration of the growth rate of the cells to be used.
- (A) is a production method including a step of adding a vascular cell and a biocompatible polymer block after forming a cell structure by the above-described method using non-vascular cells.
- the step of adding a vascular cell and a biocompatible polymer block refers to the above-described method of fusing the prepared cell structures (mosaic cell masses) with each other, and the volume under differentiation medium or growth medium. Both methods are included.
- (B) is a production method including a step of adding a non-vascular cell and a biocompatible polymer block after forming a cell structure by the above-described method using vascular cells.
- the step of adding non-vascular cells and biocompatible polymer block refers to the above-described method of fusing the prepared cell structures (mosaic cell masses) with each other under a differentiation medium or a growth medium. Both methods for increasing the volume are included.
- (C) is a production method in which a non-vascular cell and a vascular cell are used substantially simultaneously and a cell structure is formed by the method described above.
- the cell structure described above can be used in the treatment of brain damage. That is, the present invention provides a cell structure for use in the treatment of brain injury, and a therapeutic agent for brain injury comprising the cell structure described above.
- Brain damage broadly means a state in which brain function has been damaged, and examples include, but are not limited to, brain trauma, hypoxic ischemic brain injury, cerebral infarction and / or stroke. is not.
- the route of administration of the cell structure for treating brain damage and the therapeutic agent for brain injury of the present invention there are no particular limitations on the route of administration of the cell structure for treating brain damage and the therapeutic agent for brain injury of the present invention, and systemic administration (eg, parenteral administration) may be used, or local administration (eg, transplantation to a treatment site).
- systemic administration eg, parenteral administration
- local administration eg, transplantation to a treatment site.
- Administration of the cell structure for treating brain injury or the agent for treating brain injury of the present invention can be performed by various methods, for example, by injection through an injection cannula, a needle or a shunt, but is not limited thereto.
- parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intradermal administration or subcutaneous administration is preferred.
- the cell structure for brain injury treatment or the agent for treating brain injury of the present invention can also be locally administered to a treatment site (a disease site, for example, a lesion site of the brain).
- a treatment site a disease site, for example, a lesion site of the brain.
- the advantage of local administration is that cells can be more accurately targeted to the lesion site. More preferred is local administration.
- the transplantation can be performed using stereotactic surgery.
- the patient is anesthetized.
- the patient's head is placed in a nuclear magnetic resonance imaging (MRI) compatible stereotaxic frame, and a micropositioner with a microinjector is placed on the skull.
- MRI nuclear magnetic resonance imaging
- a dental drill or other suitable instrument can be used to create a burr hole in the patient's skull.
- the number of cells in the cell structure for brain injury treatment and the agent for treating brain injury of the present invention can be set to the number of cells calculated so as to produce a desired therapeutic effect.
- the number of cells per administration is preferably about 1.0 ⁇ 10 4 to 5.0 ⁇ 10 7 per kg of the patient's body weight. / Kg body weight, more preferably about 1.0 ⁇ 10 5 to 1.0 ⁇ 10 7 pieces / kg body weight, more preferably about 1.0 ⁇ 10 6 to 6.0 ⁇ 10 6 pieces / kg kg body weight.
- the treatment method of brain injury including the process of administering the cell structure for brain injury treatment of said invention to the patient who has brain injury.
- the preferred range of administration method and cell structure is the same as described above.
- the preferred range of the cell transplantation therapeutic agent and the cell structure is the same as described above.
- Example 1 Recombinant peptide (recombinant gelatin)
- CBE3 Molecular weight: 51.6 kD Structure: GAP [(GXY) 63 ] 3 G Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33% Almost 100% of amino acids are GXY repeating structures.
- the amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine.
- CBE3 has an ERGD sequence. Isoelectric point: 9.34, GRAVY value: -0.682, 1 / IOB value: 0.323
- Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 in WO2008 / 103041, except that X at the end is corrected to “P”)
- GAP GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG
- Example 2 Production of Recombinant Peptide Porous Body (Polymer Porous Body) An aluminum cylindrical cup-shaped container having a thickness of 1 mm and a diameter of 47 mm was prepared.
- the cylindrical cup has a curved surface as a side surface, the side surface is closed with 1 mm aluminum, and the bottom surface (flat plate shape) is also closed with 1 mm aluminum.
- the upper surface has an open shape.
- Teflon registered trademark
- the inner diameter of the cylindrical cup is 45 mm.
- this container is referred to as a cylindrical container.
- a CBE3 aqueous solution was prepared, and this CBE3 aqueous solution was poured into a cylindrical container.
- the CBE3 aqueous solution was cooled from the bottom using a cooling shelf in the freezer.
- the temperature of the cooling shelf, the thickness of the heat insulating plate (glass plate) sandwiched between the shelf and the cylindrical container, the final concentration of the CBE3 aqueous solution to be added, and the amount of the aqueous solution were prepared as described below.
- Condition a shelf temperature ⁇ 40 ° C., glass plate thickness 2.2 mm, final concentration of CBE3 aqueous solution 12% by mass, aqueous solution amount 4 mL.
- Condition b shelf temperature ⁇ 60 ° C., glass plate thickness 2.2 mm, final concentration of CBE3 aqueous solution 7.5% by mass, amount of aqueous solution 4 mL.
- Condition c shelf temperature ⁇ 40 ° C., glass plate thickness 2.2 mm, final concentration of CBE3 aqueous solution 4.0 mass%, amount of aqueous solution 4 mL.
- the frozen CBE3 block thus obtained was lyophilized to obtain a CBE3 porous body.
- Comparative Example 1 Production of Recombinant Peptide Simple Freezing Porous Material
- 2000 mg of CBE3 is dissolved in 18 mL of ultrapure water to prepare 20 mL of a CBE3 solution having a final concentration of 10% by mass.
- the CBE3 solution was thinly stretched to produce a thin plate-like gel having a thickness of about 4 mm.
- a white plate with a silicon frame (about 5 cm ⁇ 10 cm) attached thereto and firmly pressed so that there is no air gap was used.
- the CBE3 solution (50 ° C.) was poured into the frame. After pouring the solution, the solution was moved to 4 ° C. and gelled for about 1 hour.
- the solution was moved to ⁇ 80 ° C. and the gel was frozen for 3 hours. After freezing, it was freeze-dried with a freeze dryer (EYELA, FDU-1000).
- the freeze-dried material obtained at this time was a porous material, and the average pore size was 57.35 ⁇ m. Hereinafter, this is referred to as a simple frozen porous body.
- Example 3 Evaluation of pore size and space occupancy rate of recombinant peptide porous body
- the pore size and space occupancy were evaluated.
- the obtained porous body was thermally cross-linked at 160 ° C. for 20 hours, insolubilized, and then swollen with physiological saline for a sufficient time. Thereafter, frozen tissue sections were prepared with a microtome, and HE (hematoxylin and eosin) -stained specimens were prepared.
- a cross-sectional image with a real scale size of 1.5 mm was prepared from the sample, and the area of each hole was measured, and then the circle diameter when the area was converted into a circle was calculated to obtain the hole size.
- the average value of 20 or more holes was defined as the average hole size.
- the porous body under condition a was 66.39 ⁇ m
- the porous body under condition b was 63.17 ⁇ m
- the porous body under condition c was 56.36 ⁇ m.
- the area occupied by pores of a certain pore size was divided by the total area from the two-dimensional cross-sectional image used above, and the percentage was obtained.
- the space occupation ratio of the pores of 20 ⁇ m to 200 ⁇ m was 100% for the porous body in condition a, 99.9% for the porous body in condition b,
- the porous material of c was 99.9%.
- the space occupancy ratio of the pores of 30 ⁇ m to 150 ⁇ m was 94.2% for the porous body under condition a, 97.9% for the porous body under condition b, and 99.3% for the porous body under condition c. .
- the porous material of condition c has a bulk density of 0.05 g / cm 3 , a true density of 1.23 g / cm 3 , and a porosity of 96% (coefficient of variation (CV) value) Was 8%).
- the porous bodies of the condition a and the condition b have a porosity of 87% (CV value is 10%) and 92% (CV value is 7%), respectively.
- Example 5 Preparation of recombinant peptide block (pulverization and cross-linking of porous material)
- the CBE3 porous material obtained under the conditions a, b and c obtained in Example 2 was pulverized with a new power mill (Osaka Chemical, New Power Mill PM-2005).
- the pulverization was performed by pulverization for 5 minutes in total for 1 minute ⁇ 5 times at the maximum rotation speed.
- the obtained pulverized product was sized with a stainless steel sieve to obtain CBE3 blocks of 25 to 53 ⁇ m, 53 to 106 ⁇ m, and 106 ⁇ m to 180 ⁇ m. Thereafter, thermal crosslinking was performed at 160 ° C.
- condition a is “12% medium”
- 25 to 53 ⁇ m of condition b is “7.5% small”
- 53 to 106 ⁇ m of condition b is “7.5% medium”
- 106 to 180 ⁇ m is called “7.5% larger”
- 53 to 106 ⁇ m of condition c is called “4% medium”.
- This crosslinked gel was immersed in 1 L of 0.2M glycine solution and shaken at 40 ° C. for 2 hours. Thereafter, the cross-linked gel was washed with shaking in 5 L of ultrapure water for 1 hour, the ultrapure water was replaced with a new one, and again washed for 1 hour.
- the cross-linked gel after washing was frozen at ⁇ 80 ° C. for 5 hours, and then freeze-dried with a freeze dryer (EYELA, FDU-1000).
- the obtained freeze-dried product was pulverized with a New Power Mill (Osaka Chemical, New Power Mill PM-2005). The pulverization was performed by pulverization for 5 minutes in total for 1 minute ⁇ 5 times at the maximum rotation speed.
- the obtained pulverized product was sized with a stainless steel sieve to obtain a CBE3 / GA crosslinked ⁇ block of 25 to 53 ⁇ m.
- Comparative Example 3 Preparation of Comparative Recombinant Peptide Block
- a comparative recombinant peptide block that can be produced in a process that does not contain glutaraldehyde estimated from the prior art (Patent Document 5) is described below.
- Blocks were prepared using recombinant peptide CBE3 as a substrate.
- the simple frozen porous body obtained in Comparative Example 1 was pulverized with New Power Mill (Osaka Chemical, New Power Mill PM-2005). The pulverization was performed by pulverization for 5 minutes in total for 1 minute ⁇ 5 times at the maximum rotation speed.
- the obtained pulverized product was sized with a stainless steel sieve to obtain a CBE3 block of 25 to 53 ⁇ m. It was placed in a 160 ° C. oven and thermally crosslinked for 72 hours.
- Example 6 Measurement of tap density of recombinant peptide block
- the tap density is a value indicating how many blocks can be densely packed in a certain volume. The smaller the value, the more densely packed, that is, the block structure is complicated. It can be said that.
- the tap density was measured as follows. First, a funnel with a cap (diameter 6 mm, length 21.8 mm cylindrical: capacity 0.616 cm 3 ) was prepared, and the mass of the cap alone was measured. After that, a cap was attached to the funnel, and the block was poured from the funnel so that the block accumulated in the cap.
- the cap part was struck 200 times on a hard place such as a desk, the funnel was removed, and it was rubbed with a spatula. The mass was measured with the cap fully filled. The mass of only the block was calculated from the difference from the mass of only the cap, and the tap density was determined by dividing by the volume of the cap.
- the block of Comparative Example 2 was 715 mg / cm 3 and the block of Comparative Example 3 was 524 mg / cm 3 .
- “medium in 12%” was 372 mg / cm 3
- “7.5% small” was 213 mg / cm 3
- “medium in 7.5%” was 189 mg / cm 3
- Example 7 Calculation of “square root of area ⁇ perimeter” in a two-dimensional cross-sectional image of a recombinant peptide block
- the relationship between “square root of area” and “perimeter” of a block was determined as an index indicating the complexity of the block. . That is, it can be said that the smaller the value of “square root of area ⁇ perimeter” is, the more complicated the block is. This value was calculated using image analysis software. First, an image that shows the shape of the block was prepared. Specifically, in this example, a block group that had been well swollen with water was made into a frozen section with a microtome, and a sample stained with HE (hematoxylin and eosin)) was used.
- HE hematoxylin and eosin
- the block of Comparative Example 2 was 0.248, and the block of Comparative Example 3 was 0.139.
- “12% medium” is 0.112, “7.5% small” is 0.083, “7.5% medium” is 0.082, and “7.5% large”.
- “in 4%” was 0.061.
- the value of “square root of area ⁇ perimeter” is smaller than the block of Comparative Example 3 due to the complexity of the structure, and it is also found that there is a correlation with the tap density. It was.
- Example 8 Production of mosaic cell mass using recombinant peptide block (hMSC) Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 100,000 cells / mL with a growth medium (Takara Bio: MSCGM BulletKit (registered trademark)), and the CBE3 block prepared in Example 5 was adjusted to 0.1 mg / mL. Then, 200 ⁇ L is seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom is U-shaped), centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, allowed to stand for 24 hours, and 1 mm in diameter.
- hMSC Human bone marrow-derived mesenchymal stem cells
- a spherical cell mosaic cell mass consisting of CBE3 blocks and hMSC cells was produced (0.001 ⁇ g block per cell).
- the mosaic cell mass was spherical because it was prepared in a U-shaped plate. Also, all of “12% medium”, “7.5% small”, “7.5% medium”, “7.5% large”, and “4% medium” could be prepared in the same manner as described above.
- Example 9 Production of mosaic cell mass using recombinant peptide block (hMSC + hECFC) Human vascular endothelial progenitor cells (hECFC) were adjusted to 100,000 cells / mL with a growth medium (Lonza: EGM-2 + ECFC serum supplement), and the CBE3 block prepared in Example 5 was added to 0.05 mg / mL. After that, 200 ⁇ L was seeded on a Sumilon Celtite X96U plate, centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, and allowed to stand for 24 hours to produce a flat mosaic cell mass composed of ECFC and CBE3 blocks. .
- hMSC human bone marrow-derived mesenchymal stem cells
- a mosaic cell mass composed of hMSC, hECFC and CBE3 blocks having a spherical shape of about 1 mm in diameter was prepared. Also, all of “12% medium”, “7.5% small”, “7.5% medium”, “7.5% large”, and “4% medium” could be prepared in the same manner as described above.
- Prepared in Example 8 were Sumilon Celtite X96U Plates They were lined up and cultured for 24 hours. As a result, it was clarified that the mosaic cell mass naturally fuses when the cells arranged on the outer periphery are joined between the mosaic cell masses. Also, all of “12% medium”, “7.5% small”, “7.5% medium”, “7.5% large”, and “4% medium” could be prepared in the same manner as described above.
- Prepared in Example 9 were Sumilon Celtite X96U Plates They were lined up and cultured for 24 hours. As a result, it was clarified that the mosaic cell mass naturally fuses when the cells arranged on the outer periphery are joined between the mosaic cell masses. Also, all of “12% medium”, “7.5% small”, “7.5% medium”, “7.5% large”, and “4% medium” could be prepared in the same manner as described above.
- Example 12 In vitro ATP assay The amount of ATP (adenosine triphosphate) produced and retained by cells in each mosaic cell mass was quantified. ATP is known as an energy source for all living organisms. By quantifying the amount of ATP synthesized and retained, the state of metabolic activity and activity of cells can be known. For the measurement, CellTiter-Glo (Promega) was used. The mosaic cell masses produced in Example 8, Comparative Example 4 and Comparative Example 5 were both of Day 7, and the amount of ATP in each mosaic cell mass was quantified using CellTiter-Glo.
- ATP adenosine triphosphate
- the mosaic cell mass using the comparative CBE3 block resulted in a smaller amount of ATP.
- the mosaic cell mass using the CBE3 block for the present invention has a higher ATP amount than the mosaic cell mass using the GA cross-linked ⁇ block. This is the case when the CBE3 block for the present invention is “12% medium”, “7.5% small”, “7.5% medium”, “7.5% large”, or “4% medium”.
- more ATP was produced than the mosaic cell mass using the GA cross-linked ⁇ block (FIG. 1). That is, it became clear that the mosaic cell mass using the CBE3 block for the present invention having a complicated structure has a better survival state of the internal cells.
- Example 13 Production of Giant Mosaic Cell Mass Using Recombinant Peptide Block
- a giant mosaic cell mass by fusing 1 mm mosaic cell mass as in Example 10
- the operation can be simplified if a huge mosaic cell mass can be produced.
- 50 mL of a 1.5 mass% agarose (Agarose S) solution was added to a 9 cm Petri dish of Sumilon Celtite that had been processed without cell adhesion using a growth medium (Takara Bio: MSCGM BulletKit (registered trademark)).
- silicon having a 1 cm square shape was fixed so as to be immersed in an agarose solution by about 5 mm, and after the agarose had hardened, the silicon was removed to prepare a container having a 1 cm square recess in the agarose.
- An appropriate amount of growth medium was added thereto, and the gel was stored so as not to dry.
- the medium was removed, and a typical “in 7.5%” 16 mg of the block prepared in Example 5 and a suspension of 2.5 million cells of human bone marrow-derived mesenchymal stem cells (hMSC) were placed in the depressions. Then 25 mL of growth medium was gently added. After culturing for 1 day, a giant mosaic cell mass 1 cm square and 2-3 mm thick could be produced.
- This method can be any of the GA cross-linked ⁇ block, the comparison block and the block for the present invention, and does not depend on the type of the block.
- Example 14 Transplantation of Mosaic Cell Mass Using Recombinant Peptide Block Mice were NOD / SCID (Charles River) males, 4-6 weeks old. Under anesthesia, the mouse abdomen body hair was removed, the upper abdomen was cut subcutaneously, scissors were inserted from the cut, and the skin was peeled off from the muscles.
- Example 15 Collection of Mosaic Cell Mass Using Recombinant Peptide Block Dissection was performed 1 week and 2 weeks after transplantation. The skin of the abdomen was peeled off, and the skin to which the mosaic cell mass adhered was cut into a square size of about 1 square cm. When the mosaic cell mass was also attached to the abdominal muscle, it was collected together with the muscle.
- Example 16 Specimen analysis Tissue sections were prepared for a piece of skin to which a mosaic cell mass adhered and a mosaic cell mass before transplantation. The skin was immersed in 4% paraformaldehyde and formalin fixation was performed. Then, it embedded with paraffin and produced the tissue section
- FIG. 2 shows HE specimens two weeks after transplanting Comparative Example 6 and Comparative Example 7 in “7.5%” of Example 10.
- the survival rate described in FIG. 2 indicates the ratio of the number of living cells in the total number of cells (live cells + dead cells).
- the mosaic cell mass using the block of Comparative Example 6 (B in FIG. 2). : 37 viable cells: survival rate 45%) shows that there are few viable cells.
- the mosaic cell mass (C in FIG. 2: 116 viable cells: survival rate 84%) using the block for the present invention of Example 10 (“7.5%”) is the block of Comparative Example 6.
- the survival state of the transplanted cell between the block groups for this invention is shown in FIG.
- the survival rate described in FIG. 3 indicates the ratio of the number of living cells in the total number of cells (the number of living cells + the number of dead cells).
- the survival rate of “7.5% large” of 106-180 ⁇ m size is 57%
- the survival rate of “7.5% small” is 62%
- the survival rate of “medium 7.5%” is 84%
- the survival rate of “out of 4%” was 82%. That is, among the block groups for the present invention, “7.5% small”, “7.5% medium”, and “4% medium” are more than 106-180 ⁇ m size “7.5% large”. It was found that cell survival was improved (FIG. 3).
- FIG. 4 shows an HE specimen 2 weeks after transplantation when the mosaic cell mass of Example 10 using the block of the present invention having a size of 53 to 106 ⁇ m was transplanted. Moreover, as a result of measuring the number of blood vessels in FIG. 4, it was 63 / mm 2 . As shown in FIG. 4, it was also found that blood vessels were attracted inside the mosaic cell mass.
- Example 11 Furthermore, in the case of transplanting a mosaic cell mass containing vascular cells in a representative example of “7.5%” in Example 11, the HE specimen 2 weeks after transplantation is shown in FIG. Show. As a result of measuring the number of blood vessels in FIG. 5, it was 180 / mm 2 . Compared with the case where Example 10 is transplanted, as shown in FIG. 5, when the mosaic cell mass containing the vascular cells of Example 11 is transplanted, more blood vessels are formed inside the mosaic cell mass. It became clear.
- Example 17 Calculation of ECFC ratio and concentration in hMSC + hECFC mosaic cell cluster Among Example 9, a mosaic cell cluster using a representative "7.5% medium” was used for staining a section of a CD31 antibody for hECFC staining. Immunostaining was performed with a kit using DAB color development (EPT Anti CD31 / PECAM-1) (Daco LSAB2 kit universal K0673 Daco LSAB2 kit / HRP (DAB) for both rabbit and mouse primary antibodies). Using the image processing software ImageJ (registered trademark) and a staining method with CD31 antibody, the ratio of the area of hECFC (vascular cells) in the center was determined. The “center” here is as defined above. As a result, the ratio of the area of hECFC (vasculature cells) at the center of the representative “7.5%” mosaic cell mass of Example 9 was 99%.
- DAB color development EPT Anti CD31 / PECAM-1
- DAB DAB color development
- the above-mentioned CD31 antibody staining and HE staining are superimposed, whereby hECFC cells present in the center Density was calculated.
- the cell density of the central vascular system can be obtained by actually counting the number of cells in a sliced sample and dividing the number of cells by the volume. First, using Photoshop, the above two images were overlaid, and the number of HE-stained cell nuclei superimposed with the CD31 antibody staining was counted to calculate the number of cells.
- the volume was determined by calculating the area of the center using ImageJ (registered trademark) and multiplying the thickness of the sliced specimen by 2 ⁇ m.
- ImageJ registered trademark
- the number of hECFC (vasculature cells) at the center of the representative “7.5%” mosaic cell mass of Example 9 was 2.58 ⁇ 10 ⁇ 4 cells / ⁇ m 3 .
- Example 18 Production of Recombinant Peptide Porous Body (Polymer Porous Body) An aluminum cylindrical cup-shaped container having a thickness of 1 mm and a diameter of 47 mm was prepared.
- the cylindrical cup has a curved surface as a side surface, the side surface is closed with 1 mm aluminum, and the bottom surface (flat plate shape) is also closed with 1 mm aluminum.
- the upper surface has an open shape.
- Teflon registered trademark
- the inner diameter of the cylindrical cup is 45 mm.
- this container is referred to as a cylindrical container.
- Recombinant peptide aqueous solution was prepared with a final concentration of CBE3 of 7.5% by mass, and this recombinant peptide aqueous solution was poured into a cylindrical container.
- the aqueous recombinant peptide solution was cooled from the bottom using a cooling shelf in the freezer. At this time, the cooling process of the liquid temperature was changed by changing the temperature of the cooling shelf, the thickness of the heat insulating plate (glass plate) sandwiched between the shelf and the cylindrical container, and the amount of the aqueous solution of the recombinant peptide to be inserted.
- the shelf temperature was ⁇ 40 ° C., ⁇ 60 ° C., and ⁇ 80 ° C.
- the glass plate was 0.7 mm, 1.1 mm, and 2.2 mm
- the amount of the recombinant peptide aqueous solution was 4 mL, 12 mL, and 16 mL, and combinations thereof were performed.
- the temperature of the portion is referred to as the internal maximum liquid temperature.
- the temperature does not start to rise until the internal maximum liquid temperature reaches ⁇ 9.2 ° C., and the internal maximum liquid temperature is not frozen.
- the temperature of the solvent is equal to or lower than “solvent melting point ⁇ 3 ° C.” (FIG. 8). After this state, it can be seen that the temperature began to rise at ⁇ 9.2 ° C. and solidification heat was generated (FIG. 8). It was also confirmed that ice formation actually started at that timing. Thereafter, the temperature passes around 0 ° C. for a certain time. Here, a mixture of water and ice was present.
- the temperature starts to decrease again from 0 ° C., but at this time, the liquid portion disappears and becomes ice (FIG. 8).
- the temperature being measured is the solid temperature inside the ice. In other words, it is not liquid temperature.
- A, B, D, E, F, and H are production methods by a freezing process in which the internal maximum liquid temperature is not more than “solvent melting point ⁇ 3 ° C.” or less in an unfrozen state.
- Frozen recombinant peptide block with internal maximum liquid temperature ⁇ “solvent melting point –3 ° C” Further, this is a production method by a freezing process in which C, G and I are not frozen and the internal maximum liquid temperature does not take a liquid temperature of “solvent melting point ⁇ 3 ° C.” or lower.
- the thus obtained frozen recombinant peptide block was lyophilized to obtain a CBE3 porous material.
- A, B, D, E, F and H are derived from “CBE3 porous body with internal maximum liquid temperature ⁇ “ solvent melting point ⁇ 3 ° C. ””
- C, G and I are derived from “internal maximum liquid temperature ⁇ “ solvent melting point ⁇ It is referred to as “3 ° C. CBE3 porous body”.
- Example 19 The CBE3 porous body obtained in Example 18 was evaluated for the porous pore size and pore shape.
- the obtained porous body was subjected to thermal crosslinking at 160 ° C. for 20 hours, insolubilized, and then swelled with physiological saline for a sufficient time. Thereafter, frozen tissue sections were prepared with a microtome, and HE (hematoxylin and eosin)) stained specimens were prepared. Images of the central part of the obtained specimen are shown in FIG. 7 (internal maximum liquid temperature> “solvent melting point ⁇ 3 ° C.” and internal maximum liquid temperature ⁇ “solvent melting point ⁇ 3 ° C.”).
- the internal maximum liquid temperature is not more than “solvent melting point ⁇ 3 ° C.” in an unfrozen state, and further, “solvent melting point ⁇ 7 ° C.” It was found that by making the following, almost all the shapes of the holes can be made into spherical holes.
- a to I The pore shapes and average pore sizes for A to I are as follows.
- H Ball hole 80%, 76.58 ⁇ m
- Example 20 Preparation of recombinant peptide block (pulverization and crosslinking of porous material)
- the CBE3 porous material obtained in Example 19 was pulverized with New Power Mill (Osaka Chemical, New Power Mill PM-2005).
- the pulverization was performed by pulverization for 5 minutes in total for 1 minute ⁇ 5 times at the maximum rotation speed.
- the obtained pulverized product was sized with a stainless steel sieve to obtain recombinant peptide blocks of 25 to 53 ⁇ m and 53 to 106 ⁇ m. Thereafter, thermal crosslinking was performed at 160 ° C. under reduced pressure for 72 hours to obtain a sample.
- Example 21 For treatment of cerebral infarction: polymer solution freezing step and drying step A polytetrafluoroethylene (PTFE) cylindrical cup-shaped container having a bottom surface thickness of 3 mm, a diameter of 51 mm, a side surface thickness of 8 mm, and a height of 25 mm is used. Prepared. When the cylindrical cup has a curved surface as a side surface, the side surface is closed with 8 mm PTFE, and the bottom surface (flat plate shape) is also closed with 3 mm PTFE. On the other hand, the upper surface has an open shape. Therefore, the inner diameter of the cylindrical cup is 43 mm.
- this container is referred to as a PTFE thick / cylindrical container.
- the CBE3 aqueous solution was poured into a PTFE thick / cylindrical container, and the CBE3 aqueous solution was cooled from the bottom using a cooling shelf in a vacuum freeze dryer (TF5-85ATNNN: Takara Seisakusho).
- the combination of the container, the final concentration of the CBE3 aqueous solution, the amount of liquid, and the shelf temperature was prepared as described below.
- Example 22 For treatment of cerebral infarction: Measurement of internal maximum liquid temperature in an unfrozen state in each freezing step In the solution of Example 21, the internal maximum liquid temperature in an unfrozen state was determined in the same manner as in Example 18. It was measured. As a result, the shelf temperature is set to ⁇ 10 ° C. (before lowering to ⁇ 20 ° C.), the liquid temperature is below the melting point of 0 ° C., and freezing does not occur in this state (unfreezing / supercooling) became. Thereafter, when the shelf temperature was further lowered to ⁇ 20 ° C., the timing at which the liquid temperature suddenly rose to around 0 ° C. was confirmed, and it was found that the heat of solidification was generated and freezing was started.
- the temperature being measured is the solid temperature inside the ice, that is, not the liquid temperature (see FIG. 9).
- the maximum internal liquid temperature in the unfrozen state was ⁇ 8.8 ° C.
- the temperature at the position closest to the cooling side is defined as the cooling surface liquid temperature
- the measured cooling surface liquid temperature and internal maximum liquid temperature The differential temperature of (non-cooled surface liquid temperature) is described below.
- the differential temperature when the non-cooled surface liquid temperature reaches the melting point (0 ° C.) the differential temperature immediately before the shelf temperature is lowered from ⁇ 10 ° C. to ⁇ 20 ° C., and the differential temperature immediately before the generation of solidification heat are described.
- the “differential temperature just before” referred to in the present invention represents the highest temperature among the temperature differences that can be detected between 1 second and 20 seconds before the event.
- Example 23 For treatment of cerebral infarction: Production of polymer block (CBE3 block) from CBE3 porous body (pulverization and crosslinking of porous body)
- the CBE3 porous material obtained in Example 21 was pulverized with New Power Mill (Osaka Chemical, New Power Mill PM-2005).
- the pulverization was performed by pulverization for 5 minutes in total for 1 minute ⁇ 5 times at the maximum rotation speed.
- the obtained pulverized product was sized with a stainless steel sieve to obtain uncrosslinked blocks of 25 to 53 ⁇ m, 53 to 106 ⁇ m, and 106 ⁇ m to 180 ⁇ m. Thereafter, thermal crosslinking was performed at 160 ° C.
- crosslinking time was 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, and 96 hours
- crosslinking time was 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, and 96 hours
- Example 24 For treatment of cerebral infarction: Tap density of CBE3 block, For the CBE3 block (53 to 106 ⁇ m) produced in Example 23, the tap density was measured in the same manner as in Example 6. As a result, the CBE3 block of Example 23 was 135 mg / cm 3 . From this, it can be seen that the CBE3 block of Example 23 satisfies that the tap density is 10 mg / cm 3 or more and 500 mg / cm 3 or less as in Examples 12 and 20.
- Example 25 For treatment of cerebral infarction: Calculation of “square root of area ⁇ perimeter” in two-dimensional cross-sectional image of CBE3 block The CBE3 block (53 to 106 ⁇ m) prepared in Example 23 was treated in the same manner as Example 7. The value of “square root of area ⁇ perimeter” was measured. As a result, the block of Example 23 was 0.053. From this, the CBE3 block of Example 23 satisfies that the value of the square root of the cross-sectional area divided by the circumference is 0.01 or more and 0.13 or less in Examples 12 and 20. I understand.
- Example 26 Production of mosaic cell mass using CBE3 block (GFP expressing rat MSC) GFP (green fluorescent protein) -expressing rat bone marrow-derived mesenchymal stem cells (GFP rat MSC: Fischer 344 (F344) Rat Mesenchymal Stem Cells with GFP, CSC-C1313, Creative Bioarray) to 100,000 cells / mL in recommended growth medium
- GFP rat MSC green fluorescent protein
- F344 Rat Mesenchymal Stem Cells with GFP, CSC-C1313, Creative Bioarray
- Example 27 Production of mosaic cell mass using CBE3 block (SD rat bone marrow cells)
- Example 23 SD (Sprague-Dawley) rat bone marrow cells (rat BMSC, BMC01, Cosmo Bio) were adjusted to 150,000 cells / mL with a recommended growth medium (Cosmo Bio: culture medium for bone marrow cells, BMCM).
- the CBE3 block (53-106 ⁇ m) prepared in step 1 was added to 0.15 mg / mL.
- Example 28 Production of mosaic cell mass using CBE3 block (hMSC) Human bone marrow-derived mesenchymal stem cells (hMSC) were adjusted to 100,000 cells / mL with a growth medium (Takara Bio: MSCGM BulletKit (registered trademark)), and the CBE3 block prepared in Example 23 (53-106 ⁇ m) was 0. .1 mg / mL was added. 200 ⁇ L of the obtained mixture was seeded on a Sumilon Celtite X96U plate (Sumitomo Bakelite, bottom U-shaped), centrifuged (600 g, 5 minutes) with a desktop plate centrifuge, allowed to stand for 24 hours, and spherical with a diameter of 1 mm.
- a growth medium Tea Bio: MSCGM BulletKit (registered trademark)
- a mosaic cell mass consisting of CBE3 block and hMSC cells was prepared (0.001 ⁇ g block per cell).
- the mosaic cell mass was spherical because it was prepared in a U-shaped plate.
- This mosaic cell mass is the same as the mosaic cell mass using D in Example 20 in the same in vitro assay as in Example 12 and the results of transplantation into animals by the same evaluation as in Examples 14 to 16. High performance (high cell viability) was shown.
- Example 8 Cell-containing CBE3 sponge
- the CBE3 porous material obtained in Example 21 was molded into a diameter of 5 mm x 1 mm to obtain a CBE3 sponge.
- GFP-expressing rat bone marrow-derived mesenchymal stem cells (GFP rat MSC: Fischer 344 (F344) Rat Mesenchymal Stem Cells with GFP, CSC-C1313, Creative Bioarray) are suspended in the recommended medium. Cells were grown to 0.5 ⁇ 10 6 cells / cell by seeding 1.2 ⁇ 10 4 cells / cell and culturing for 24 days. Thereby, a cell-containing CBE3 sponge was obtained.
- Example 29 Preparation of rat cerebral infarction animal model (MCAO model SD rat) For SD rat males 9 weeks old, ⁇ Neurosurgery.68 (6): 1733-1742, June 2011, T Sugiyama et.al.Therapeutic Impact of Human Bone Marrow Stromal Cells Expanded by Animal Serum-Free Medium for Cerebral Infarct in A cerebral infarction was created in the right brain by performing middle cerebral artery occlusion (MCAO) in the same manner as that of Rats. Under isoflurane anesthesia, the rat right temporal region was opened, the skull was opened, and the right cerebral middle cerebral artery was ligated with a suture.
- MCAO middle cerebral artery occlusion
- the blood vessels in the field of view were cauterized with a bipolar core regulator and closed. Thereafter, the carotid artery was temporarily ligated for 90 minutes, and blood flow was resumed to produce a cerebral infarction model.
- motor function evaluation it evaluated by quantifying the motor function evaluation by Rotarod, or the improvement of the left turning action. Rotarod's evaluation was set to accelerate to 4-40 rpm in 300 seconds, and six tests a day were conducted at intervals of 5 minutes or more each time. The average of these 6 tests was evaluated as a percentage (%) of the normal value of the same individual (100% if normal).
- the left-turning behavior is based on the fact that the lower function of the left body is observed because the MCAO model creates a cerebral infarction in the right brain. Specifically, it was evaluated in which direction the rat first walked with only the front legs of the rat on the ground and the back legs floating in the air. The direction was left: -1 point, straight: 0 point, right: 0 point, and evaluated by quantifying the average value obtained by performing the test six times. The normal rat goes straight, so it becomes 0 point, and the rat that made the right cerebral infarction moves counterclockwise, so a value close to -1 point comes out.
- Example 30 Preparation of rat cerebral infarction animal model (MCAO model Nude rat) A cerebral infarction in the right brain, which was subjected to middle cerebral artery occlusion (MCAO), was prepared in the same manner as in Example 29 for 9 weeks old male immunodeficient nude rats. In this motor function evaluation, the left turning behavior was evaluated in the same manner as in Example 29.
- MCAO model Nude rat Middle cerebral artery occlusion
- Example 31 Administration of GFP-expressing rat MSC mosaic cell mass, cell suspension, or cell-containing CBE3 sponge to cerebral infarction rats After the onset of the MCAO model SD rats prepared in Example 29 ( 7 days after treatment with MCAO) On day 7, the GFP-expressing rat MSC mosaic cell mass produced in Example 26, the cell suspension in the same cell suspended in 100 ⁇ L of PBS as a comparative example, and the cell produced in Comparative Example 8 The contained CBE3 sponge was administered as a local administration by being directly implanted near the site of brain injury.
- Example 32 Improvement of motor function of cerebral infarction rat (GFP expressing rat MSC mosaic cell mass) As a result of Example 31, as shown in FIG. 10, in the state where the GFP-expressing rat MSC was used as a cell, in the mosaic cell mass (administered on the seventh day in the graph), 14 days and 21 days were very prominent. It was found that significant improvement in motor function was observed. On the other hand, in the cell suspension administration used as a comparison, no improvement in motor function was confirmed even after administration. Similarly, the cell-containing CBE3 sponge for comparison did not show a significant effect like a mosaic cell mass, and the motor function remained flat.
- the GFP-expressing rat MSC used for transplantation is a Fischer rat cell and the administered cerebral infarction rat is an SD rat, it was found that the effect of treating cerebral infarction can also be obtained with other (allogeneic) cells.
- Example 33 Administration of mosaic cell mass of SD rat bone marrow cells or cell suspension to cerebral infarction rat 7 days after onset (after MCAO treatment) for MCAO model SD rat prepared in Example 29
- the SD rat bone marrow cell mosaic cell mass produced in Example 27, or a cell suspension in which the same cells are suspended in 100 ⁇ L of PBS as a comparative example is locally administered and injected directly into the vicinity of the brain injury site with a syringe.
- ⁇ 10 6 cells / animal 1.33 ⁇ 10 6 cells / kg body weight
- cell suspensions were 1.6 ⁇ 10 6 cells / animal and 0.38 ⁇ 10 6 cells / animal, administered between administration groups
- Comparative evaluation was performed with the number of cells changed in a state in which the number of cells was combined. The number of animals was 10 in each group.
- Example 34 Improvement of motor function of cerebral infarction rat (SD rat bone marrow cell mosaic cell mass) As a result of Example 33, as shown in FIG. 11, in the state where SD rat bone marrow cells were used as cells, in the mosaic cell mass (administered on the seventh day in the graph), 21 days, 28 days, and 35 days were obtained. It was found that there was a very significant improvement in motor function of left-turning behavior. In addition, the effect of improvement was higher in the mosaic cell mass having a larger number of cells. On the other hand, in the cell suspension administration used as a comparison, the improvement of motor function was not observed at all in the group of few cells (0.38 ⁇ 10 6 cells / animal).
- Example 36 Improvement of motor function in cerebral infarction rat (hMSC mosaic cell mass) As a result of Example 35, as shown in FIG. 11, in the state where hMSC was used as the cells, the mosaic cell mass was administered after 14 days (administered on the 7th day in the graph), 14 days, 21 days, 28 days, 35 days. It was found that there was a very significant improvement in motor function of left-turning behavior. From this, it became clear that the effect can be confirmed in the state using human cells. At the same time, since the cells used for transplantation were human cells and the transplanted side was a rat, it was found that the treatment of cerebral infarction can be obtained even if heterogeneous cells are used in an immunocompromised state.
- Example 37 hMSC expression analysis The hMSC used in Example 28 was analyzed for gene expression in the nervous system. The RT-PCR method was used for the analysis. Primers were purchased from TaqMan® Gene Expression Assays from life technologies applied biosystems. Analyzed subjects were Sox2 (Cat. # 4331182 Hs01053049_s1 Amplicon Length: 91), Nestin (Nestin, Cat. # 4331182 Hs0418731_g1 Amplicon Length: 581) GAD1 (GABA synthesis, Cat. # 4331182 Hs01065893_m1 Amplicon Length: 100), GRIA1 (Glutamate receptor 1, Cat.
- HTR1B Serotonin Receptor 1B, Cat. # 4331182 Hs00265286_s1 Amplicon Length: 67
- HTR2A Serotonin Receptor 2A, Cat. # 4331182 Hs01035li_T1 , Cat. # 4331182 Hs00984349_m1 Amplicon Length: 58
- Ascl1 nodegenerative stem cell neuron differentiation marker, Cat. # 4351372 Hs04187546_g1 Amplicon Length: 81
- Hes1 Neural stem cell astrocyte differentiation marker, Cat. # 4331182 Hs00172878_m1 Amplicon Length: 78
- Olig2 nodegenerative stem cell oligodendrocyte differentiation marker, Cat. It is.
- RT-PCR normal concentration RT-PCR (RT-PCR was performed using 200 ng of extracted RNA) and high concentration RT-PCR (RT using PCR of 1000 ng of extracted RNA) -PCR was performed) and the magnitude relationship of expression was examined.
- RT-PCR was performed under the following conditions. RNA extraction from the cells was performed according to the protocol using an extraction kit NucleoSpin RNA XS (MACHEREY-NAGEL # U0902A).
- the RT-PCR reagent kit is PrimeScript One Step RT-PCR Kit ver. 2 (Dye Plus) (TaKaRa PR057A) was used.
- the thermal cycler conditions for RT-PCR were 50 cycles at 50 ° C. for 30 minutes and 94 ° C. for 2 minutes, followed by “94 ° C. 30 seconds, 60 ° C. 30 seconds, 72 ° C. 15 seconds” as one cycle.
- Example 38 In Examples 26, 27 and 28, the mosaic cell mass was formed by allowing the CBE3 block and the cells to stand for 24 hours. In Example 38, the time required for formation of the mosaic cell mass was examined under the same conditions as in Example 28 using the same CBE3 block and cells as in Example 28. Using the U-shaped plate, the process of forming the cell structure (mosaic cell mass) was confirmed with a microscope. Initially, CBE3 blocks and cells are dispersed, and the results of measuring the length of the spread of the dispersion are shown in FIG. The diameter of the vertical axis in FIG. 12 indicates the length of spread between the block and the cell. As the cell structure is formed, the length (diameter) of the spread between the block and the cell becomes shorter.
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Abstract
Description
(1) 生体親和性高分子ブロックと、少なくとも一種類の細胞とを含み、複数個の上記細胞間の隙間に複数個の上記生体親和性高分子ブロックが配置されている脳損傷治療用細胞構造体であって、上記生体親和性高分子ブロックのタップ密度が10mg/cm3以上500mg/cm3以下であるか、又は上記生体親和性高分子ブロックの二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下である、脳損傷治療用細胞構造体。
(2) 上記細胞が、少なくとも間葉系幹細胞及び/又は骨髄細胞を含む、(1)に記載の脳損傷治療用細胞構造体。
(3) 1回の投与当たり投与される細胞数が、1.0×105 ~ 1.0×107 個/kg体重である、(1)又は(2)に記載の脳損傷治療用細胞構造体。
(4) 脳損傷が、脳外傷、低酸素性虚血性脳損傷、脳梗塞及び/又は脳卒中である、(1)から(3)の何れかに記載の脳損傷治療用細胞構造体。
(5) 上記生体親和性高分子ブロック一つの大きさが10μm以上300μm以下である、(1)から(4)の何れかに記載の脳損傷治療用細胞構造体。
(6) 上記細胞構造体の厚さ又は直径が400μm以上3cm以下である、(1)から(5)の何れかに記載の脳損傷治療用細胞構造体。
(7) 上記細胞構造体が、細胞1個当り0.0000001μg以上1μg以下の生体親和性高分子ブロックを含む、(1)から(6)の何れかに記載の脳損傷治療用細胞構造体。
(8) 上記生体親和性高分子ブロックがリコンビナントペプチドからなる、(1)から(7)の何れかに記載の脳損傷治療用細胞構造体。
(9) 上記リコンビナントペプチドが、
配列番号1に記載のアミノ酸配列からなるペプチド;
配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ生体親和性を有するペプチド;又は
配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有するアミノ酸配列からなり、かつ生体親和性を有するペプチド;
の何れかである、(8)に記載の脳損傷治療用細胞構造体。
(10) 上記生体親和性高分子ブロックにおいて、上記生体親和性高分子が熱、紫外線又は酵素により架橋されている、(1)から(9)の何れかに記載の脳損傷治療用細胞構造体。
(12) 上記生体親和性高分子ブロックが、
生体親和性高分子の溶液を、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融点より3℃低い温度以下となる、凍結処理により凍結する工程a;及び
上記工程aで得られた凍結した生体親和性高分子を凍結乾燥する工程b:
を含む方法により製造される生体親和性高分子ブロックである、(1)から(11)の何れかに記載の脳損傷治療用細胞構造体。
(13) 上記生体親和性高分子ブロックが、
生体親和性高分子の溶液を、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融点より3℃低い温度以下となる、凍結処理により凍結する工程a;
上記工程aで得られた凍結した生体親和性高分子を凍結乾燥する工程b;及び
上記工程bで得られた多孔質体を粉砕する工程c:
を含む方法により製造される生体親和性高分子ブロックである、(1)から(12)の何れかに記載の脳損傷治療用細胞構造体。
(14) 上記工程aにおいて、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融より7℃低い温度以下となる、(12)又は(13)に記載の脳損傷治療用細胞構造体。
(15) 上記細胞構造体が、上記生体親和性高分子ブロックと上記細胞とを混合し、10時間以上培養することにより得られる細胞構造体である、(1)から(14)の何れかに記載の脳損傷治療用細胞構造体。
(16) (1)から(15)の何れかに記載の脳損傷治療用細胞構造体を複数個融合することにより得られる、脳損傷治療用細胞構造体。
(17) タップ密度が10mg/cm3以上500mg/cm3以下であるか、又は二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下である生体親和性高分子ブロックと細胞とを混合し、10時間以上培養する工程を含む、(1)から(16)の何れかに記載の脳損傷治療用細胞構造体の製造方法。
(18) (1)から(16)の何れかに記載の脳損傷治療用細胞構造体を含む、脳損傷治療剤。
(20) 脳損傷治療剤の製造のための、(1)から(16)の何れかに記載の脳損傷治療用細胞構造体の使用。
本発明は、生体親和性高分子ブロックと、少なくとも一種類の細胞とを含み、複数個の上記細胞間の隙間に複数個の上記生体親和性高分子ブロックが配置されている脳損傷治療用細胞構造体であって、上記生体親和性高分子ブロックのタップ密度が10mg/cm3以上500mg/cm3以下であるか、又は上記生体親和性高分子ブロックの二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下である、脳損傷治療用細胞構造体、その製造方法、並びに上記脳損傷治療用細胞構造体を含む脳損傷治療剤に関する。なお、本発明における細胞構造体は、本明細書中において、モザイク細胞塊(モザイク状になっている細胞塊)と称する場合もある。
(1-1)生体性親和性高分子
生体性親和性とは、生体に接触した際に、長期的かつ慢性的な炎症反応などのような顕著な有害反応を惹起しないことを意味する。本発明で用いる生体親和性高分子は、生体に親和性を有するものであれば、生体内で分解されるか否かは特に限定されないが、生分解性高分子であることが好ましい。非生分解性材料として具体的には、ポリテトラフルオロエチレン(PTFE)、ポリウレタン、ポリプロピレン、ポリエステル、塩化ビニル、ポリカーボネート、アクリル、ステンレス、チタン、シリコーンおよびMPC(2-メタクリロイルオキシエチルホスホリルコリン)などが挙げられる。生分解性材料としては、具体的にはリコンビナントペプチドなどのポリペプチド(例えば、以下に説明するゼラチン等)、ポリ乳酸、ポリグリコール酸、乳酸・グリコール酸コポリマー(PLGA)、ヒアルロン酸、グリコサミノグリカン、プロテオグリカン、コンドロイチン、セルロース、アガロース、カルボキシメチルセルロース、キチン、およびキトサンなどが挙げられる。上記の中でも、リコンビナントペプチドが特に好ましい。これら生体親和性高分子には細胞接着性を高める工夫がなされていてもよい。具体的には、1.「基材表面に対する細胞接着基質(フィブロネクチン、ビトロネクチン、ラミニン)や細胞接着配列(アミノ酸一文字表記で現わされる、RGD配列、LDV配列、REDV配列、YIGSR配列、PDSGR配列、RYVVLPR配列、LGTIPG配列、RNIAEIIKDI配列、IKVAV配列、LRE配列、DGEA配列、及びHAV配列)ペプチドによるコーティング」、「基材表面のアミノ化、カチオン化」、又は「基材表面のプラズマ処理、コロナ放電による親水性処理」といった方法を使用できる。
本発明で用いる生体親和性高分子は、架橋されているものでもよいし、架橋されていないものでもよいが、架橋されているものが好ましい。架橋されている生体親和性高分子を使用することにより、培地中で培養する際および生体に移植した際に瞬時に分解してしまうことを防ぐという効果が得られる。一般的な架橋方法としては、熱架橋、アルデヒド類(例えば、ホルムアルデヒド、グルタルアルデヒドなど)による架橋、縮合剤(カルボジイミド、シアナミドなど)による架橋、酵素架橋、光架橋、紫外線架橋、疎水性相互作用、水素結合、イオン性相互作用などが知られている。本発明ではグルタルアルデヒドを使用しない架橋方法を使用することが好ましい。本発明では、アルデヒド類又は縮合剤を使用しない架橋方法を使用することがより好ましい。即ち、本発明における生体親和性高分子ブロックは、好ましくは、グルタルアルデヒドを含まない生体親和性高分子ブロックであり、より好ましくは、アルデヒド類又は縮合剤を含まない生体親和性高分子ブロックである。本発明で使用する架橋方法としては、さらに好ましくは熱架橋、紫外線架橋、又は酵素架橋であり、特に好ましくは熱架橋である。
本発明で言うリコンビナントゼラチンとは、遺伝子組み換え技術により作られたゼラチン類似のアミノ酸配列を有するポリペプチドもしくは蛋白様物質を意味する。本発明で用いることができるリコンビナントゼラチンは、コラーゲンに特徴的なGly-X-Yで示される配列(X及びYはそれぞれ独立にアミノ酸の何れかを示す)の繰り返しを有するものが好ましい。ここで、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよい。好ましくは、細胞接着シグナルが一分子中に2配列以上含まれている。本発明で用いるリコンビナントゼラチンとしては、コラーゲンの部分アミノ酸配列に由来するアミノ酸配列を有するリコンビナントゼラチンを用いることができる。例えばEP1014176、US特許6992172号、国際公開WO2004/85473、国際公開WO2008/103041等に記載のものを用いることができるが、これらに限定されるものではない。本発明で用いるリコンビナントゼラチンとして好ましいものは、以下の態様のリコンビナントゼラチンである。
この最小アミノ酸配列の含有量は、細胞接着・増殖性の観点から、タンパク質1分子中3~50個が好ましく、さらに好ましくは4~30個、特に好ましくは5~20個である。最も好ましくは12個である。
好ましくは、リコンビナントゼラチンはテロペプタイドを有さない。
好ましくは、リコンビナントゼラチンは、アミノ酸配列をコードする核酸により調製された実質的に純粋なポリペプチドである。
(1)配列番号1に記載のアミノ酸配列からなるペプチド;
(2)配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ生体親和性を有するペプチド;又は
(3)配列番号1に記載のアミノ酸配列と80%以上(さらに好ましくは90%以上、特に好ましくは95%以上、最も好ましくは98%以上)の配列同一性を有するアミノ酸配列からなり、かつ生体親和性を有するペプチド;
本発明では、上記した生体親和性高分子からなるブロック(塊)を使用する。
本発明における生体親和性高分子ブロックの形状は特に限定されるものではない。例えば、不定形、球状、粒子状、粉状、多孔質状、繊維状、紡錘状、扁平状およびシート状であり、好ましくは、不定形、球状、粒子状、粉状および多孔質状であり、より好ましくは不定形である。不定形とは、表面形状が均一でないもののことを示し、例えば、岩のような凹凸を有する物を示す。
生体親和性高分子ブロックの製造方法は、上記(1-4)に記載した条件を満たす生体親和性高分子ブロックが得られる限りは特に限定されない。例えば、生体親和性高分子の多孔質体を、粉砕機(ニューパワーミルなど)を用いて粉砕することにより顆粒の形態とすることができ、これにより上記(1-4)に記載した条件を満たす生体親和性高分子ブロックを得ることができる。
生体親和性高分子の溶液を、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融点より3℃低い温度(「溶媒融点-3℃」)以下となる、凍結処理により凍結する工程a;及び
上記工程aで得られた凍結した生体親和性高分子を凍結乾燥する工程b:
を含む方法により、生体親和性高分子の多孔質体を製造することができる。上記工程によれば、空孔の内、球孔の占める割合が50%以上とすることができるからである。
より好ましくは、上記工程aにおいて、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融より7℃低い温度(「溶媒融点-7℃」)以下となる凍結処理により凍結することができる。この工程によれば、空孔の内、球孔の占める割合が80%以上とすることができるからである。
多孔質体の空孔サイズ30μm~150μmが占める空間占有率は、好ましくは60%以上100%以下であり、より好ましくは70%以上100%以下、さらに好ましくは80%以上100%以下、特に好ましくは90%以上100%以下である。
本発明で用いる細胞は、脳損傷の治療を行えるものであれば任意の細胞を使用することができ、その種類は特に限定されない。また、使用する細胞は1種でもよいし、複数種の細胞を組合せて用いてもよい。また、使用する細胞として、好ましくは、動物細胞であり、より好ましくは脊椎動物由来細胞、特に好ましくはヒト由来細胞である。脊椎動物由来細胞(特に、ヒト由来細胞)の種類は、万能細胞、体性幹細胞、前駆細胞、又は成熟細胞の何れでもよい。万能細胞としては、例えば、胚性幹(ES)細胞、生殖幹(GS)細胞、又は人工多能性幹(iPS)細胞を使用することができる。体性幹細胞としては、例えば、間葉系幹細胞(MSC)、造血幹細胞、羊膜細胞、臍帯血細胞、骨髄細胞(骨髄由来細胞)、心筋幹細胞、脂肪由来幹細胞、又は神経幹細胞を使用することができる。前駆細胞及び成熟細胞としては、例えば、神経、脳、又は骨髄に由来する細胞を使用することができる。ヒト由来細胞としては、例えば、ES細胞、iPS細胞、MSC、神経細胞、血管内皮細胞、骨髄由来細胞、又は造血幹細胞を使用することができる。また、細胞の由来は、自家細胞又は他家細胞の何れでも構わない。例えば、脳虚血・脳梗塞に対しては、神経前駆細胞、または、神経細胞に分化可能な細胞を投与することができる。
神経系の遺伝子としては、Sox2, Nestin(ネスチン), NeuroD1(Neurogenic differentiation 1), GAD1(GABA合成), GRIA1(グルタミン酸受容体1),GRIA2(グルタミン酸受容体2), CHRM1(アセチルコリン受容体1), GABRA1(GABAA受容体α1), GABBR1(GABAB受容体1), CHAT(アセチルコリン合成), DDC(セロトニン/DOPA合成),HTR1A(セロトニン受容体1A), HTR1B(セロトニン受容体1B)、HTR2A(セロトニン受容体2A), 5-HTT(セロトニントランスポーター), Ascl1(神経幹細胞ニューロン分化マーカー), Hes1(神経幹細胞アストロサイト分化マーカー), 及びOlig2(神経幹細胞オリゴデンドロサイト分化マーカー)などが挙げられるが、特に限定されない。本発明で用いる細胞としては、上記した神経系の遺伝子のうちの1種以上、好ましくは2種以上、より好ましくは3種以上、さらに好ましくは5種以上、さらに好ましくは7種以上、特に好ましくは10種以上、最も好ましくは13種以上を発現している細胞(好ましくはMSC)を用いることができる。
本発明においては、生体親和性高分子ブロックと細胞とを用いて、複数個の細胞間の隙間に複数個の生体親和性高分子ブロックをモザイク状に3次元的に配置させることによって細胞移植のために適した厚みを有することが可能となる。さらに、生体親和性高分子ブロックと細胞とがモザイク状に3次元に配置されることにより、構造体中で細胞が均一に存在する細胞構造体を形成され、外部から細胞構造体の内部への栄養送達を可能となる。これにより、本発明の脳損傷治療用細胞構造体を用いて、細胞移植を行うと、移植された細胞の壊死を抑制し、移植が可能となる。なお、ここでいう「壊死の抑制」とは、細胞構造体とせず、細胞のみを移植した場合と比較して、壊死の程度が低いことを意味する。
本発明の脳損傷治療用細胞構造体としては、細胞構造体の内部において血管形成されているものも好ましい。
本発明の脳損傷治療用細胞構造体は、生体親和性高分子ブロックと、少なくとも一種類の細胞とを混合することによって製造することができる。より具体的には、本発明の脳損傷治療用細胞構造体は、生体親和性高分子ブロックと、細胞とを交互に配置することにより製造できる。製造方法は特に限定されないが、好ましくは生体親和性高分子ブロックを形成したのち、細胞を播種する方法である。具体的には、生体親和性高分子ブロックと細胞含有培養液との混合物をインキュベートすることによって、本発明の脳損傷治療用細胞構造体を製造することができる。例えば、容器中、容器に保持される液体中で、細胞と、予め作製した生体親和性高分子ブロックとをモザイク状に配置する。配置の手段としては、自然凝集、自然落下、遠心、攪拌を用いることで、細胞と生体親和性基材からなるモザイク状の配列形成を促進又は制御することが好ましい。
(a)別々に調製した細胞構造体(モザイク細胞塊)同士を融合させる、又は
(b)分化培地又は増殖培地下でボリュームアップさせる、
などの方法により所望の大きさの細胞構造体を製造することができる。融合の方法、ボリュームアップの方法は特に限定されない。
(a)は非血管系細胞を用いて前述の方法で細胞構造体を形成した後、血管系細胞および生体親和性高分子ブロックを加える工程を有する製造方法である。ここで、「血管系細胞および生体親和性高分子ブロックを加える工程」とは、前述した、調製した細胞構造体(モザイク細胞塊)同士を融合させる方法、および、分化培地又は増殖培地下でボリュームアップさせる方法、いずれも含むものである。
(b)は血管系細胞を用いて前述の方法で細胞構造体を形成した後、非血管系細胞および生体親和性高分子ブロックを加える工程を有する製造方法である。ここで、「非血管系細胞および生体親和性高分子ブロックを加える工程」とは、前述した、調製した細胞構造体(モザイク細胞塊)同士を融合させる方法、および、分化培地又は増殖培地下でボリュームアップさせる方法、いずれも含むものである。
(c)は、非血管系細胞および血管系細胞を実質的に同時に使用し、前述の方法で細胞構造体を形成させる製造方法である。
上記した細胞構造体は、脳損傷の治療において使用することができる。即ち、本発明は、脳損傷の治療において使用するための細胞構造体、並びに上記した細胞構造体を含む脳損傷治療剤を提供するものである。
脳損傷とは、脳の機能に損傷が生じた状態を広く意味し、例えば、脳外傷、低酸素性虚血性脳損傷、脳梗塞及び/又は脳卒中などが挙げられるが、これらに限定されるものではない。
さらに本発明によれば、脳損傷治療剤の製造のための、上記した本発明の脳損傷治療用細胞構造体の使用が提供される。細胞移植治療剤及び細胞構造体の好適な範囲は前述と同様である。
リコンビナントペプチド(リコンビナントゼラチン)として以下記載のCBE3を用意した(WO2008/103041に記載)。
CBE3
分子量:51.6kD
構造: GAP[(GXY)63]3G
アミノ酸数:571個
RGD配列:12個
イミノ酸含量:33%
ほぼ100%のアミノ酸がGXYの繰り返し構造である。CBE3のアミノ酸配列には、セリン、スレオニン、アスパラギン、チロシン及びシステインは含まれていない。CBE3はERGD配列を有している。
等電点:9.34、GRAVY値:-0.682、1/IOB値:0.323
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G
厚さ1mm、直径47mmのアルミ製円筒カップ状容器を用意した。円筒カップは曲面を側面としたとき、側面は1mmのアルミで閉鎖されており、底面(平板の円形状)も1mmのアルミで閉鎖されている。一方、上面は開放された形をしている。また、側面の内部にのみ、肉厚1mmのテフロン(登録商標)を均一に敷き詰め、結果として円筒カップの内径は45mmになっている。以後、この容器のことを円筒形容器と呼称する。
条件a:棚板温度-40℃、硝子板の厚さ2.2mm、CBE3水溶液の最終濃度12質量%、水溶液量4mL。
条件b:棚板温度-60℃、硝子板の厚さ2.2mm、CBE3水溶液の最終濃度7.5質量%、水溶液量4mL。
条件c:棚板温度-40℃、硝子板の厚さ2.2mm、CBE3水溶液の最終濃度4.0質量%、水溶液量4mL。
50℃で、2000mgのCBE3を18mLの超純水に溶解し、終濃度10質量%のCBE3溶液を20mL作製する。そのCBE3溶液を薄く伸ばして4mm厚程度の薄い板状のゲルを作製した。容器については、白い板に、シリコン枠(5cm×10cm程度)をつけて、空気の隙間がないようにしっかりとシリコン枠を押し付けたものを使用した。上記の枠内へ上記のCBE3溶液(50℃)を流し込んだ。液を流し込んだら、4℃へ移して約1時間ゲル化させ、固まっていることを確認した後、-80℃へ移して、ゲルを3時間凍結させた。凍結後、凍結乾燥機(EYELA、FDU-1000)で凍結乾燥を行った。尚、この時に得られた凍結乾燥体は多孔質体ではあり、平均ポアサイズが57.35μmとなっていた。以後、これを単純凍結多孔質体と呼称する。
実施例2で得られたCBE3多孔質体および比較例1で得られた単純凍結多孔質体について、多孔質体の空孔サイズと空間占有率の評価を実施した。得られた多孔質体を160℃で20時間の熱架橋を施し、不溶化したのち、十分時間、生理食塩水で膨潤した。その後、ミクロトームで凍結組織切片を作製し、HE(ヘマトキシリン・エオシン)染色標本を作製した。 標本から実スケール1.5mm大の断面像を用意し、個々の空孔面積を計測し、その後、上記面積を円換算した場合の円直径を算出し、空孔サイズとした。この空孔の20ヶ以上の平均値を平均空孔サイズとした。その結果、条件aの多孔質体は66.39μm、条件bの多孔質体は63.17μm、条件cの多孔質体は56.36μmであった。
実施例2で得られたCBE3多孔質体について、空孔率を測定した。測定に当たっては、嵩密度(ρ)と真密度(ρc)を測定し、空孔率(P = 1-ρ/ρc(%))を求めた。CBE3多孔質体の嵩密度(ρ)は、乾燥質量と体積から算出した。真密度(ρc)は、ハバード型形の比重瓶法により求めた。サンプル数(N)=4の結果として、条件cの多孔質体では嵩密度が0.05g/cm3、真密度が1.23g/cm3、空孔率96%(変動係数(CV)値は8%)であることが明らかになった。また、条件a及び条件bの多孔質体ではそれぞれ空孔率が87%(CV値は10%)及び92%(CV値は7%)であることが分かった。
実施例2で得られた条件a、条件b及び条件cのCBE3多孔質体をニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm、53~106μm、106μm~180μmのCBE3ブロックを得た。 その後、減圧下160℃で熱架橋(架橋時間は24時間、48時間、56時間、60時間、72時間、84時間、96時間、120時間、288時間の9種類を実施した)を施して、試料を得た。以下、条件aの53~106μmを「12%中」、条件bの25~53μmを「7.5%小」、条件bの53~106μmを「7.5%中」、条件bの106~180μmを「7.5%大」、条件cの53~106μmを「4%中」と呼ぶ。
特許文献5に記載されているグルタルアルデヒドを使用する比較例として、基材としてリコンビナントペプチドCBE3を用いて、不定形のGA架橋μブロックを作製した。1000mgのCBE3を9448μLの超純水に溶解し、1mol/LのHClを152μL添加後、終濃度1.0質量%となるように、25質量%グルタルアルデヒドを400μL添加し、50℃で3時間反応させ、架橋ゲルを作製した。この架橋ゲルを、1Lの0.2Mグリシン溶液へ浸漬し、40℃2時間振とうさせた。その後、架橋ゲルを、5Lの超純水中で1時間振とう洗浄、超純水を新しい物へ置換し、再び洗浄1時間、を繰り返し、計6回洗浄した。洗浄後の架橋ゲルを、-80℃で5時間凍結させた後、凍結乾燥機(EYELA、FDU-1000)で凍結乾燥を行った。得られた凍結乾燥体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μmのCBE3・GA架橋μブロックを得た。
比較例として、従来技術(特許文献5)から類推されるグルタルアルデヒドを含まない工程で作製した場合にできる比較用リコンビナントペプチドブロックを、以下に記載の通り作製した。基材としてリコンビナントペプチドCBE3を用いてブロックを作製した。比較例1で得られた単純凍結多孔質体を、ニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μmのCBE3ブロックを得た。それを160℃のオーブンに入れ、72時間熱架橋した。
タップ密度は、ある体積にどれくらいのブロックを密に充填できるかを表す値であり、値が小さいほど、密に充填できない、すなわちブロックの構造が複雑であると言える。タップ密度は、以下のように測定した。まず、ロートの先にキャップ(直径6mm、長さ21.8mmの円筒状:容量0.616cm3)が付いたものを用意し、キャップのみの質量を測定した。その後、ロートにキャップを付け、ブロックがキャップに溜まるようにロートから流し込んだ。十分量のブロックを入れた後、キャップ部分を200回、机などの硬いところにたたきつけ、ロートをはずし、スパチュラですりきりにした。このキャップにすりきり一杯入った状態で質量を測定した。キャップのみの質量との差からブロックのみの質量を算出し、キャップの体積で割ることで、タップ密度を求めた。
一方、実施例5のブロックは「12%中」が372mg/cm3、「7.5%小」が213mg/cm3、「7.5%中」が189mg/cm3、「7.5%大」が163mg/cm3、「4%中」が98mg/cm3であった。実施例5のブロックでは、構造の複雑性に由来して、比較例3のブロックに対して、タップ密度は小さくなることが分かった。
ブロックの複雑性を示す指標として、ブロックの「面積の平方根」と「周囲長」の関係を求めた。すなわち、ブロックの「面積の平方根÷周囲長」の値が小さい方がより複雑であると言える。この値は、画像解析ソフトを用いて算出した。まず、ブロックの形が分かる画像を用意した。具体的に、本実施例では、水で良く膨潤させたブロック群を、ミクロトームで凍結切片にし、HE(ヘマトキシリン・エオシン))染色した標本を用いた。ブロック以外に細胞などが存在する場合は、photoshop(登録商標)で、自動選択ツールで製剤のみを抽出し、画像上にブロックのみとなるようにする。その画像を、Imagej(登録商標)を用いて、ブロックの面積と周囲長を求め、「面積の平方根÷周囲長」の値を算出した。ただし、10μm以下のブロックは除いた。
一方、実施例5のブロックは、「12%中」が0.112、「7.5%小」が0.083、「7.5%中」が0.082、「7.5%大」が0.071、「4%中」が0.061であった。実施例5のブロックでは、構造の複雑性に由来して、比較例3のブロックに対して、「面積の平方根÷周囲長」の値は小さくなり、また、タップ密度と相関があることも分かった。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))にて10万cells/mLに調整し、実施例5で作製したCBE3ブロックを0.1mg/mLとなるように加えた後、200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mm程度の球状の、CBE3ブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は球状であった。また、これは「12%中」、「7.5%小」、「7.5%中」、「7.5%大」、「4%中」のいずれも上記と同様に作成できた。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))にて10万cells/mLに調整し、比較例3で作製したCBE3ブロックを0.1mg/mLとなるように加えた後、200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mm程度の球状の、CBE3ブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は、球状であった。
比較用のグルタルアルデヒドを含むモザイク細胞塊を以下の通り作成した。ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))にて10万cells/mLに調整し、比較例2で作製したGA架橋μブロックを0.1mg/mLとなるように加えた後、200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mm程度の球状の、GA架橋μブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は、球状であった。
ヒト血管内皮前駆細胞(hECFC)を増殖培地(Lonza:EGM-2+ECFC serum supplement)にて10万cells/mLに調整し、実施例5で作製したCBE3ブロックを0.05mg/mLとなるように加えた後、200μLをスミロンセルタイトX96Uプレートに播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、扁平状の、ECFCとCBE3ブロックからなるモザイク細胞塊を作製した。その後、培地を除去し、ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))にて10万cells/mLに調整し、実施例5で作製したCBE3ブロックを0.1mg/mLとなるように加えた後、hECFCモザイク細胞塊がある200μLをスミロンセルタイトX96Uプレートに播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mm程度の球状の、hMSCとhECFCとCBE3ブロックからなるモザイク細胞塊を作製した。また、これは「12%中」、「7.5%小」、「7.5%中」、「7.5%大」、「4%中」のいずれも上記と同様に作成できた。
実施例8で作製した2日目のモザイク細胞塊(本発明用のCBE3ブロックを使用)5個をスミロンセルタイトX96Uプレート中で並べ、24時間培養を行った。その結果、モザイク細胞塊同士の間を、外周部に配された細胞が結合させることで、モザイク細胞塊が自然に融合することが明らかになった。また、これは「12%中」、「7.5%小」、「7.5%中」、「7.5%大」、「4%中」のいずれも上記と同様に作成できた。
実施例9で作製した2日目のモザイク細胞塊(本発明用のCBE3ブロックを使用)5個をスミロンセルタイトX96Uプレート中で並べ、24時間培養を行った。その結果、モザイク細胞塊同士の間を、外周部に配された細胞が結合させることで、モザイク細胞塊が自然に融合することが明らかになった。また、これは「12%中」、「7.5%小」、「7.5%中」、「7.5%大」、「4%中」のいずれも上記と同様に作成できた。
比較例4で作製した2日目のモザイク細胞塊(比較用のCBE3ブロック由来)5個をスミロンセルタイトX96Uプレート中で並べ、24時間培養を行った。その結果、モザイク細胞塊同士の間を、外周部に配された細胞が結合させることで、モザイク細胞塊が自然に融合することが明らかになった。
比較例5で作製した2日目のモザイク細胞塊(GA架橋μブロック由来)5個をスミロンセルタイトX96Uプレート中で並べ、24時間培養を行った。その結果、モザイク細胞塊同士の間を、外周部に配された細胞が結合させることで、モザイク細胞塊が自然に融合することが明らかになった。
各モザイク細胞塊中の細胞が産生・保持しているATP(アデノシン三リン酸)量を定量した。ATPは生物全般のエネルギー源として知られ、ATP合成量・保持量を定量することで、細胞の代謝活性の状態、活動状態を知ることができる。測定には、CellTiter-Glo(Promega社)を用いた。実施例8および比較例4、比較例5で作製したモザイク細胞塊について、ともにDay7のもので、CellTiter-Gloを用いて、各モザイク細胞塊中のATP量を定量した。その結果、GA架橋μブロックを用いたモザイク細胞塊に比べ、比較用のCBE3ブロックを用いたモザイク細胞塊では、ATP量が少ない結果となった。一方、本発明用のCBE3ブロックを用いたモザイク細胞塊は、GA架橋μブロックを用いたモザイク細胞塊よりもATP量が多いことが分かった。これは本発明用のCBE3ブロックが、「12%中」、「7.5%小」、「7.5%中」、「7.5%大」、「4%中」いずれの場合においても、同様に、GA架橋μブロックを用いたモザイク細胞塊よりも多くのATPを産生している結果となった(図1)。つまり、複雑な構造を持つ本発明用のCBE3ブロックを用いたモザイク細胞塊の方が、内部の細胞の生存状態が良好であることが明らかになった。
同様に、GA架橋の比較例2の場合、「面積の平方根÷周囲長」が0.248で性能が得られていたが、GA(グルタルアルデヒド)を用いない比較例3では0.139でも性能が得られなかった。一方で、実施例5の「12%中」:0.112、「7.5%小」:0.083、「7.5%中」:0.082、「7.5%大」:0.071、「4%中」:0.061で性能が得られていることから、GAを用いない場合では、特に「面積の平方根÷周囲長」が0.13以下であることが重要だとわかる。
実施例10のように1mmのモザイク細胞塊を融合して、巨大なモザイク細胞塊を作製することは可能であるが、一度に巨大なモザイク細胞塊を作製できる方が、操作を簡便化できる。ここで、細胞の接着しない加工を施したスミロンセルタイトの9cmシャーレに、を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))で1.5質量%アガロース(Agarose S)溶液を50mL入れた。その際、1cm四方の棒状にしたシリコンを5mm程度アガロース溶液中に浸るように固定し、アガロースが固まった後、シリコンを取り除き、アガロース中に1cm四方の窪みができた容器を作成した。そこに増殖培地を適量入れ、ゲルが乾かないように保存した。培地を取り除き、実施例5で作製したブロックの内、代表的な「7.5%中」16mgと、250万cellsのヒト骨髄由来間葉系幹細胞(hMSC)の懸濁物を窪みに入れ、その後静かに25mLの増殖培地を添加した。1日培養後、1cm四方、厚さ2-3mmの巨大モザイク細胞塊を作製できた。これを、25mLの増殖培地を入れたスミロンセルタイトの9cmシャーレに移し、さらに2日培養後、25mLの増殖培地を入れたスピナーフラスコに移して攪拌培養し、培養7日後のモザイク細胞塊の断面のHE染色標本を作製した。その結果、培養7日後でも内部の細胞が生存していることを確認した。このように、細胞とブロックを混合し、型に流して培養することで、巨大なモザイク細胞塊を作製可能であることが明らかになった。また、この方法は、GA架橋μブロック、比較用ブロック及び本発明用のブロックの何れでも可能であり、ブロックの種類に依存しない。
マウスはNOD/SCID(チャールズリバー)のオス、4~6週齢のものを用いた。麻酔下でマウス腹部の体毛を除去し、上腹部の皮下に切れ込みを入れ、切れ込みからはさみを差し込み、皮膚を筋肉からはがした後、実施例10、実施例11、比較例6、及び比較例7で作成したモザイク細胞塊をピンセットですくい、切れ込みから1.5cmほど下腹部寄りの皮下に移植し、皮膚の切れ込み部を縫合した。
解剖は移植から1週後及び2週後に行った。腹部の皮膚をはがし、モザイク細胞塊が付着した皮膚を、約1平方cmの正方形の大きさに切り取った。モザイク細胞塊が腹部の筋肉にも付着している場合は、筋肉と共に採取した。
モザイク細胞塊が付着した皮膚片および、移植前のモザイク細胞塊について組織切片を作製した。皮膚を4%パラホルムアルデヒドに浸漬し、ホルマリン固定を行った。その後、パラフィンで包埋し、モザイク細胞塊を含む皮膚の組織切片を作製した。切片はHE染色(ヘマトキシリン・エオシン染色)を行った。
比較例7のGA架橋μブロックを用いたモザイク細胞塊(図2のA:生細胞数100個:生存率80%)に比べ、比較例6のブロックを用いたモザイク細胞塊(図2のB:生細胞数37個:生存率45%)では、生細胞が少ないことが分かる。一方、実施例10の本発明用のブロック(「7.5%中」)を用いたモザイク細胞塊(図2のC:生細胞数116個:生存率84%)は、比較例6のブロックを用いたモザイク細胞塊(図2のB:生細胞数37個:生存率45%)に比べて、生細胞が多く、生存が良好であることが分かった。この結果は、実施例12でのin vitroアッセイの結果とも一致し、本発明用のブロックを採用することによって、移植した細胞の生存を良くすることが可能であることが分かった。
106~180μmサイズの「7.5%大」の生存率は57%であり、「7.5%小」の生存率は62%であり、「7.5%中」の生存率は84%であり、「4%中」の生存率は82%であった。即ち、本発明用のブロック群内間では、106~180μmサイズの「7.5%大」よりも、「7.5%小」、「7.5%中」、「4%中」が更に細胞の生存が良くなることが分かった(図3)。また、最上の移植結果をもたらすものとしては、「7.5%中」と「4%中」が、「7.5%小」よりも、移植細胞の生存が良いことも分かった(図3)。即ち、高分子ブロックのタップ密度又は高分子ブロックの二次元断面像における断面積の平方根÷周囲長の値が所定の範囲内となるような構造を有することが重要であり、中でも、「53~106μm」>「25~53μm」>「106~180μm」という順で移植後の細胞生存が良くなることも分かった。
実施例9の内、代表的な「7.5%中」を用いたモザイク細胞塊は、切片をhECFC染色用にCD31抗体(EPT Anti CD31/PECAM-1)にDAB発色を用いたキット(ダコLSAB2キット ユニバーサル K0673 ダコLSAB2キット/HRP(DAB) ウサギ・マウス一次抗体両用)による免疫染色を行った。画像処理ソフトImageJ(登録商標)、CD31抗体による染色方法を使用し、中心部のhECFC(血管系の細胞)の面積の割合を求めた。なお、ここでいう「中心部」とは上記定義したものである。
その結果、実施例9の代表的な「7.5%中」モザイク細胞塊の中心部のhECFC(血管系の細胞)の面積の割合は99%であった。
その結果、実施例9の代表的な「7.5%中」モザイク細胞塊の中心部のhECFC(血管系の細胞)の細胞数は2.58×10-4cells/μm3であった。
厚さ1mm、直径47mmのアルミ製円筒カップ状容器を用意した。円筒カップは曲面を側面としたとき、側面は1mmのアルミで閉鎖されており、底面(平板の円形状)も1mmのアルミで閉鎖されている。一方、上面は開放された形をしている。また、側面の内部にのみ、肉厚1mmのテフロン(登録商標)を均一に敷き詰め、結果として円筒カップの内径は45mmになっている。以後、この容器のことを円筒形容器と呼称する。
A 棚板温度-40℃で硝子板2.2mm、液量4mLは、-9.2℃
B 棚板温度-40℃で硝子板1.1mm、液量4mLは、-8.3℃
C 棚板温度-40℃で硝子板0.7mm、液量4mLは、-2.2℃
D 棚板温度-60℃で硝子板2.2mm、液量4mLは、-7.2℃
尚、ここでいうDが実施例2でいうところの「条件b」である。
E 棚板温度-80℃で硝子板2.2mm、液量4mLは、-3.9℃
F 棚板温度-80℃で硝子板1.1mm、液量4mLは、-3.1℃
G 棚板温度-80℃で硝子板0.7mm、液量4mLは、 5.8℃
H 棚板温度-40℃で硝子板2.2mm、液量12mLは、-6.5℃
I 棚板温度-40℃で硝子板2.2mm、液量16mLは、-2.4℃
また、C、G及びIが未凍結状態で内部最高液温が「溶媒融点-3℃」以下の液温をとらない凍結工程による製造法である。(内部最高液温>「溶媒融点-3℃」の凍結リコンビナントペプチドブロック)
実施例18で得られたCBE3多孔質体について、多孔質の空孔サイズと空孔形状の評価を実施した。得られた多孔質体を160℃、20時間の熱架橋を施し、不溶化したのち、十分時間、生理食塩水で膨潤。その後、ミクロトームで凍結組織切片を作製し、HE(ヘマトキシリン・エオシン))染色標本を作製した。
得られた標本の中心部分の画像を図7(内部最高液温>「溶媒融点-3℃」、と、内部最高液温≦「溶媒融点-3℃」)に示した。その結果、内部最高液温>「溶媒融点-3℃」(C,G,I)では、空孔は80%以上が柱/平板孔となり、球孔は20%以下であった。一方、内部最高液温≦「溶媒融点-3℃」(A,B,D,E,F,H)では、空孔は50%以上が球孔となっていた。また、この内部最高液温≦「溶媒融点-7℃」(A,B,D)では、空孔は80%以上が球孔となっており、ほぼ全てが球孔で構成されていた。これにより、多孔質体の空孔形状を球孔にするには、未凍結状態で内部最高液温が「溶媒融点-3℃」以下となることが重要で、更に「溶媒融点-7℃」以下とすることで、空孔の形状をほぼ全て球孔にすることができることが分かった。
A:球孔100%、62.74μm
B:球孔100%、65.36μm
C:柱/平板孔90%、79.19μm
D:球孔100%、63.17μm
E:球孔70%、69.44μm
F:球孔50%、53.98μm
G:柱/平板孔90%、79.48μm
H:球孔80%、76.58μm
I:柱/平板孔90%、79.65μm
実施例19で得られたCBE3多孔質体をニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm及び53~106μmのリコンビナントペプチドブロックを得た。その後、減圧下160℃で72時間、熱架橋を施して、試料を得た。これらの試料は、タップ密度が10mg/cm3以上500mg/cm3以下であること、又は上記高分子ブロックの二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下であることを充足するものであった。これらの試料を使用して実施例8及び実施例10と同じようにして作製したモザイク細胞塊ではA~Iによらず、実施例12と同じin vitroアッセイ、並びに実施例14~16と同じ評価による動物への移植結果において、いずれも、比較用ブロックよりも高い性能(細胞の高い生存率)を示した。ただ、これらのA~Iの中で性能差を見ると僅かな差が生じており、A、B、Dが最も性能が高く、ついでE、F、H、その後にC、G、Iがくるという順番になっていた。つまり、これは多孔質体の空孔形状によって、性能差を生じうることを示している。実施例16では、代表的な結果としてD(実施例2でいうところの「条件b」)について詳細に記述している。
底面厚さ3mm、直径51mm、側面厚さ8mm、高さ25mmのポリテトラフルオロエチレン(PTFE)製円筒カップ状容器を用意した。円筒カップは曲面を側面としたとき、側面は8mmのPTFEで閉鎖されており、底面(平板の円形状)も3mmのPTFEで閉鎖されている。一方、上面は開放された形をしている。よって、円筒カップの内径は43mmになっている。以後、この容器のことをPTFE厚・円筒形容器と呼称する。
PTFE厚・円筒形容器、CBE3水溶液の最終濃度4質量%、水溶液量8mL。
棚板温度の設定は、-10℃になるまで冷却し、-10℃で1時間、その後-20℃で2時間、さらに-40℃で3時間、最後に-50℃で1時間凍結を行った。本凍結品はその後、棚板温度を-20℃設定に戻してから-20℃で24時間の真空乾燥を行い、24時間後にそのまま真空乾燥を続けた状態で棚板温度を20℃へ上昇させ、十分に真空度が下がる(1.9×105Pa)まで、さらに20℃で48時間の真空乾燥を実施した後に、真空凍結乾燥機から取り出した。それによって多孔質体を得た。
実施例21の溶液内で、実施例18と同様にして未凍結状態での内部最高液温を測定した。その結果、棚板温度-10℃設定区間(-20℃度に下げる前)において液温が融点である0℃を下回り、かつその状態で凍結が起こっていない(未凍結・過冷却)状態となった。その後、棚板温度を-20℃へ更に下げていくことによって、液温が0℃付近へ急激に上昇するタイミングが確認され、ここで凝固熱が発生し凍結が開始されたことが分かった。また、そのタイミングで実際に氷形成が始まっていることも確認出来た。その後、温度は0℃付近を一定時間経過していく。ここでは、水と氷の混合物が存在する状態となっていた。最後0℃から再び温度降下が始まるが、この時、液体部分はなくなり氷となっている。従って、測定している温度は氷内部の固体温度となり、つまり液温ではなくなる(図9を参照)。
一方、水面付近の内部最高液温(非冷却面液温)に対して、冷却側に最も近い位置の温度を冷却面液温、と定義し測定した場合の冷却面液温と内部最高液温(非冷却面液温)の差温を以下に記す。
非冷却面液温が融点(0℃)になった時の差温、棚板温度を-10℃から-20℃へ下げる直前の差温と、凝固熱発生直前の差温を記載する。尚、本発明で言うところの「直前の差温」とは、上記イベントの1秒前~20秒前までの間で検知可能な温度差の内、最も高い温度のことを表している。
非冷却面液温が融点(0℃)になった時の差温:1.1℃
-10℃から-20℃へ下げる直前の差温:0.2℃
凝固熱発生直前の差温:1.1℃
(図9を参照)
実施例21で得られたCBE3多孔質体をニューパワーミル(大阪ケミカル、ニューパワーミルPM-2005)で粉砕した。粉砕は、最大回転数で1分間×5回、計5分間の粉砕で行った。得られた粉砕物について、ステンレス製ふるいでサイズ分けし、25~53μm、53~106μm、106μm~180μmの未架橋ブロックを得た。その後、減圧下160℃で熱架橋(架橋時間は8時間、16時間、24時間、48時間、72時間、96時間の6種類を実施した)を施して、試料CBE3ブロックを得た。以下、48時間架橋を施した。尚、架橋時間の違いは本願の評価においては性能に影響が見られない為、ここでは48時間架橋したものを代表として使用している。
実施例23で作製したCBE3ブロック(53~106μm)について、実施例6と同様にして、タップ密度を測定した。その結果、実施例23のCBE3ブロックは135mg/cm3であった。このことから実施例23のCBE3ブロックは実施例12及び実施例20でいうところの、タップ密度が10mg/cm3以上500mg/cm3以下であることを充足するものであることが分かる。
実施例23で作製したCBE3ブロック(53~106μm)について、実施例7と同様にして、「面積の平方根÷周囲長」の値を測定した。その結果、実施例23のブロックは0.053であった。このことから実施例23のCBE3ブロックは実施例12及び実施例20でいうところの、断面積の平方根÷周囲長の値が0.01以上0.13以下であることを充足するものであることが分かる。
GFP(緑色蛍光タンパク質)発現ラット骨髄由来間葉系幹細胞(GFPラットMSC:Fischer 344 (F344) Rat Mesenchymal Stem Cells with GFP、CSC-C1313、Creative Bioarray)を推奨増殖培地にて10万cells/mLに調整し、実施例23で作製したCBE3ブロック(53~106μm)を0.1mg/mLとなるように加えた。得られた混合物200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mmの球状の、CBE3ブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は球状であった(GFP発現ラットMSCモザイク細胞塊、と呼称する)。尚、本モザイク細胞塊は、実施例12と同じin vitroアッセイ、並びに実施例14~16と同じ評価による動物への移植結果において、いずれも、実施例20のDを用いたモザイク細胞塊と同程度の高い性能(細胞の高い生存率)を示した。
SD(Sprague-Dawley)ラット骨髄細胞(ラットBMSC、BMC01、コスモバイオ社)を推奨増殖培地(コスモバイオ社:骨髄細胞用培養メディウム、BMCM)にて15万cells/mLに調整し、実施例23で作製したCBE3ブロック(53-106μm)を0.15mg/mLとなるように加えた。得られた混合物200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mmの球状の、CBE3ブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は球状であった。尚、本モザイク細胞塊は、実施例12と同じin vitroアッセイ、並びに実施例14~16と同じ評価による動物への移植結果において、いずれも、実施例20のDを用いたモザイク細胞塊と同程度の高い性能(細胞の高い生存率)を示した。
ヒト骨髄由来間葉系幹細胞(hMSC)を増殖培地(タカラバイオ:MSCGM BulletKit(登録商標))にて10万cells/mLに調整し、実施例23で作製したCBE3ブロック(53-106μm)を0.1mg/mLとなるように加えた。得られた混合物200μLをスミロンセルタイトX96Uプレート(住友ベークライト、底がU字型)に播種し、卓上プレート遠心機で遠心(600g、5分)し、24時間静置し、直径1mmの球状の、CBE3ブロックとhMSC細胞からなるモザイク細胞塊を作製した(細胞1個当たり0.001μgのブロック)。なお、U字型のプレート中で作製したため、本モザイク細胞塊は球状であった。尚、本モザイク細胞塊は、実施例12と同じin vitroアッセイ、並びに実施例14~16と同じ評価による動物への移植結果において、いずれも、実施例20のDを用いたモザイク細胞塊と同程度の高い性能(細胞の高い生存率)を示した。
実施例21で得られたCBE3多孔質体をφ5mm×1mmに成型し、CBE3スポンジを得た。このCBE3スポンジに対して、GFP発現ラット骨髄由来間葉系幹細胞(GFPラットMSC:Fischer 344 (F344) Rat Mesenchymal Stem Cells with GFP、CSC-C1313、Creative Bioarray)を、推奨培地に懸濁した状態で1.2×104cells/個を播種し、24日間培養することで0.5×106cells/個まで細胞を増殖させた。これにより細胞含有CBE3スポンジを得た。
SDラット雄9週令に対して、「Neurosurgery. 68(6):1733-1742, June 2011, T Sugiyama et. al. Therapeutic Impact of Human Bone Marrow Stromal Cells Expanded by Animal Serum-Free Medium for Cerebral Infarct in Rats」の方法と同様にして、中大脳動脈閉塞術(MCAO)を施すことで右脳に脳梗塞を作製した。イソフルラン麻酔下で、ラット右側側頭部を開頭、頭蓋骨をあけて右脳側中大脳動脈を縫合糸で結紮した。視野内の血管をバイポーラコアレユレータで焼灼し、閉頭した。その後、頚動脈を90分間一時結紮し、血流を再開させることで、脳梗塞モデルを作製した。
なお、運動機能評価としては、Rotarodによる運動機能評価、または左旋回行動の改善を定量化することで評価した。Rotarodの評価は、4-40rpmへ300秒で加速していく設定とし、1日6回の試験を各回5分以上の間隔をあけて実施した。この6回の試験の平均値を、同個体の正常時の数値に対する割合(%)として評価した(正常ならば100%となる)。
免疫不全ヌード(Nude)ラット雄9週令に対して、実施例29と同様にして、中大脳動脈閉塞術(MCAO)を施した右脳の脳梗塞を作製した。この運動機能評価も実施例29と同様の方法で左旋回行動の評価を行った。
実施例29で作製したMCAOモデルSDラットに対して、発症後(MCAO処置後)7日の時に、実施例26で作製したGFP発現ラットMSCモザイク細胞塊、また比較例として同じ細胞をPBS100μLに懸濁した状態の細胞懸濁液、また比較例8で作製した細胞含有CBE3スポンジを局所投与として、脳損傷部位付近に直接埋殖することにより投与した。
投与した量は、GFP発現ラットMSCモザイク細胞塊は25個/匹(細胞数では0.5×106 cells/匹=1.34×106cells/kg体重)、細胞懸濁液は0.5×106cells/匹、細胞含有CBE3スポンジは1個/匹(細胞数では0.5×106cells/匹)とし、投与群間で投与された細胞数を合わせた状態にて比較評価した。尚、動物の個体数は各群10匹で実施した。
実施例31の結果、図10に示す通り、GFP発現ラットMSCを細胞として用いた状態で、モザイク細胞塊では投与後(グラフで7日目に投与)に、14日、21日と非常に顕著な運動機能の改善が認められることが分かった。一方、比較として用いた細胞懸濁液投与では、投与後も運動機能の改善は全く確認されなかった。同様に、比較である細胞含有CBE3スポンジでもモザイク細胞塊のような顕著な効果は確認されず、運動機能は横ばいのままであった。尚、モザイク細胞塊、及び細胞懸濁液の投与については、同じ実験を2回行っている為、グラフ中に「2回目」というデータを示している。
このことから、運動機能の改善効果の大きさは、「モザイク細胞塊」≫「細胞含有スポンジ」≧「細胞懸濁液」となることが分かった。また、梗塞発生から7日目という急性期において使用しても効果が得られることが分かった。これは48時間以内の超急性期でなくても効果が得られることを意味している。さらに、移植に用いたGFP発現ラットMSCはFischerラット細胞であり、投与された脳梗塞ラットはSDラットであることから、他家(同種)細胞でも脳梗塞治療効果が得られることが分かった。
実施例29で作製したMCAOモデルSDラットに対して、発症後(MCAO処置後)7日の時に、実施例27で作製したSDラット骨髄細胞モザイク細胞塊、また比較例として同じ細胞をPBS100μLに懸濁した状態の細胞懸濁液を局所投与として、脳損傷部位付近に直接注射器で注入することにより投与した。
投与した量は、SDラット骨髄細胞モザイク細胞塊は53個/匹、13個/匹(細胞数では1.6×106cells/匹=4.35×106cells/kg体重、0.38×106cells/匹=1.33×106cells/kg体重)、細胞懸濁液は1.6×106cells/匹、0.38×106cells/匹とし、投与群間で投与された細胞数を合わせた状態にて、細胞数を変えた比較評価を行った。尚、動物の個体数は各群10匹で実施した。
実施例33の結果、図11に示す通り、SDラット骨髄細胞を細胞として用いた状態で、モザイク細胞塊では投与後(グラフで7日目に投与)に、21日、28日、35日と非常に顕著な左旋回行動の運動機能改善が認められる、と分かった。また、改善の効果は細胞数が多いモザイク細胞塊の方が高かった。 一方、比較として用いた細胞懸濁液投与では、少ない細胞数(0.38×106cells/匹)群では全く運動機能の改善が見られなかった。また、多い細胞数(1.6×106cells/匹)を投与しても、細胞懸濁液群では、運動機能改善には限界があり、同数をモザイク細胞塊で投与した郡より顕著に改善が低いことはもとより、少ない細胞数のモザイク細胞塊群よりも運動機能改善が悪いことが明らかとなった。
実施例30で作製したMCAOモデルNudeラット(免疫不全)に対して、発症後(MCAO処置後)7日の時に、実施例28で作製したhMSCモザイク細胞塊を局所投与として、脳損傷部位付近に直接埋殖することにより投与した。投与した量は、hMSCモザイク細胞塊は45個/匹(細胞数では0.89×106cells/匹=5.50×106cells/kg体重)とした。尚、動物の個体数は10匹で実施した。
実施例35の結果、図11に示す通り、hMSCを細胞として用いた状態で、モザイク細胞塊では投与後(グラフで7日目に投与)に、14日、21日、28日、35日と非常に顕著な左旋回行動の運動機能改善が認められる、と分かった。
このことから、ヒトの細胞を用いた状態で、効果が確認できることが明らかとなった。同時に、移植に用いた細胞がヒト細胞であり、移植された側がラットであることから、免疫不全状態においては、異種細胞を用いても脳梗塞治療効果が得られることが分かった。
実施例28にて使用したhMSCについて、神経系の遺伝子発現を解析した。解析にはRT-PCR手法を用いた。プライマーはlife technologies applied biosystemsのTaqMan(登録商標) Gene Expression Assaysから購入した。解析した対象は、Sox2(Cat.#4331182 Hs01053049_s1 Amplicon Lenghe:91), Nestin(ネスチン、Cat.#4331182 Hs04187831_g1 Amplicon Lenghe:58), NeuroD1(Neurogenic differentiation 1、Cat.#4331182 Hs01922995_s1 Amplicon Lenghe:110), GAD1(GABA合成、Cat.#4331182 Hs01065893_m1 Amplicon Lenghe:100), GRIA1(グルタミン酸受容体1、Cat.#4331182 Hs00181348_s1 Amplicon Lenghe:86),GRIA2(グルタミン酸受容体2、Cat.#4331182 Hs00181331_m1 Amplicon Lenghe:71), CHRM1(アセチルコリン受容体1、Cat.#4331182 Hs00265195_s1 Amplicon Lenghe:82), GABRA1(GABAA受容体α1、Cat.#4331182 Hs00971228_m1 Amplicon Lenghe:82), GABBR1(GABAB受容体1、Cat.#4331182 Hs00559488_m1 Amplicon Lenghe:68), CHAT(アセチルコリン合成、Cat.#4331182 Hs00252848_m1 Amplicon Lenghe:64), DDC(セロトニン/DOPA合成、Cat.#4351372 Hs01105048_m1 Amplicon Lenghe:70),HTR1A(セロトニン受容体1A、Cat.#4331182 Hs00265014_s1 Amplicon Lenghe:75), HTR1B(セロトニン受容体1B、Cat.#4331182 Hs00265286_s1 Amplicon Lenghe:67)、HTR2A(セロトニン受容体2A、Cat.#4331182 Hs01033524_m1 Amplicon Lenghe:99), 5-HTT(セロトニントランスポーター、Cat.#4331182 Hs00984349_m1 Amplicon Lenghe:58), Ascl1(神経幹細胞ニューロン分化マーカー、Cat.#4351372 Hs04187546_g1 Amplicon Lenghe:81), Hes1(神経幹細胞アストロサイト分化マーカー、Cat.#4331182 Hs00172878_m1 Amplicon Lenghe:78), 及びOlig2(神経幹細胞オリゴデンドロサイト分化マーカー、Cat.#4331182 Hs00300164_s1 Amplicon Lenghe:86)である。
細胞からのRNA抽出は抽出キットNucleoSpin RNA XS(MACHEREY-NAGEL社#U0902A)を使用し、プロトコール通りに実施した。RT-PCRの試薬キットはPrimeScript One Step RT-PCR Kit ver.2(Dye Plus)(TaKaRa PR057A)を用いた。RT-PCRのサーマルサイクラー条件は50℃で30分、94℃で2分処理した後、「94℃30秒、60℃30秒、72℃15秒」を1サイクルとして、50サイクル実施した。
++:通常PCR、高濃度PCRともに標的遺伝子を検出できた場合。
+:通常PCRでは補足出来ないが、高濃度PCRで標的遺伝子を検出できた場合。
-:通常PCRでも高濃度PCRでも標的遺伝子を検出できなかった場合。
尚、検出出来たかどうかは、電気泳動によりバンドが目視確認出来たかどうかで判断した。
実施例26、27及び28においては、モザイク細胞塊は、CBE3ブロックと細胞とを24時間静置することにより形成させた。本実施例38では、実施例28と同様のCBE3ブロックと細胞とを用いて実施例28と同様の条件において、モザイク細胞塊の形成に必要な時間を調べた。U字型プレートで使用して、細胞構造体(モザイク細胞塊)が形成される過程を顕微鏡で確認した。最初はCBE3ブロックと細胞とが分散しているが、その分散の広がりの長さを測定した結果を図12に示す。図12の縦軸の直径は、ブロックと細胞との広がりの長さを示す。細胞構造体を形成するにつれてブロックと細胞との広がりの長さ(直径)は短くなる。図12の結果から分かるように、CBE3ブロックと細胞を混合して、1、2、3、4、5、6及び7時間静置した状態では、本発明で言うモザイク細胞塊はできておらず、細胞とブロックとが別々の状態で存在していた。即ち、図12において、1、2、3、4、5、6及び7時間静置した状態では、ブロックと細胞との広がりの長さが減少していることから、塊が一つにまとまっていないことが分かる。一方、15、18、21、23、26、29、45及び69時間静置した状態では、ブロックと細胞との広がり(直径)の長さが変化しなくなっていることから、一つのモザイク細胞塊の形成が終わっていたことが分かる。
Claims (18)
- 生体親和性高分子ブロックと、少なくとも一種類の細胞とを含み、複数個の前記細胞間の隙間に複数個の前記生体親和性高分子ブロックが配置されている脳損傷治療用細胞構造体であって、前記生体親和性高分子ブロックのタップ密度が10mg/cm3以上500mg/cm3以下であるか、又は前記生体親和性高分子ブロックの二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下である、脳損傷治療用細胞構造体。
- 前記細胞が、少なくとも間葉系幹細胞及び/又は骨髄細胞を含む、請求項1に記載の脳損傷治療用細胞構造体。
- 1回の投与当たり投与される細胞数が、1.0×105 ~ 1.0×107 個/kg体重である、請求項1又は2に記載の脳損傷治療用細胞構造体。
- 脳損傷が、脳外傷、低酸素性虚血性脳損傷、脳梗塞及び/又は脳卒中である、請求項1から3の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記生体親和性高分子ブロック一つの大きさが10μm以上300μm以下である、請求項1から4の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記細胞構造体の厚さ又は直径が400μm以上3cm以下である、請求項1から5の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記細胞構造体が、細胞1個当り0.0000001μg以上1μg以下の生体親和性高分子ブロックを含む、請求項1から6の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記生体親和性高分子ブロックがリコンビナントペプチドからなる、請求項1から7の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記リコンビナントペプチドが、
配列番号1に記載のアミノ酸配列からなるペプチド;
配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ生体親和性を有するペプチド;又は
配列番号1に記載のアミノ酸配列と80%以上の配列同一性を有するアミノ酸配列からなり、かつ生体親和性を有するペプチド;
の何れかである、請求項8に記載の脳損傷治療用細胞構造体。 - 前記生体親和性高分子ブロックにおいて、前記生体親和性高分子が熱、紫外線又は酵素により架橋されている、請求項1から9の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記生体親和性高分子ブロックが、生体親和性高分子の多孔質体を粉砕することにより得られる顆粒の形態にある、請求項1から10の何れか一項に記載の脳損傷治療用細胞構造体。
- 前記生体親和性高分子ブロックが、
生体親和性高分子の溶液を、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融点より3℃低い温度以下となる、凍結処理により凍結する工程a;及び
前記工程aで得られた凍結した生体親和性高分子を凍結乾燥する工程b:
を含む方法により製造される生体親和性高分子ブロックである、請求項1から11の何れか一項に記載の脳損傷治療用細胞構造体。 - 前記生体親和性高分子ブロックが、
生体親和性高分子の溶液を、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融点より3℃低い温度以下となる、凍結処理により凍結する工程a;
前記工程aで得られた凍結した生体親和性高分子を凍結乾燥する工程b;及び
前記工程bで得られた多孔質体を粉砕する工程c:
を含む方法により製造される生体親和性高分子ブロックである、請求項1から12の何れか一項に記載の脳損傷治療用細胞構造体。 - 前記工程aにおいて、溶液内で最も液温の高い部分の液温である内部最高液温が、未凍結状態で、溶媒融より7℃低い温度以下となる、請求項12又は13に記載の脳損傷治療用細胞構造体。
- 前記細胞構造体が、前記生体親和性高分子ブロックと前記細胞とを混合し、10時間以上培養することにより得られる細胞構造体である、請求項1から14の何れか一項に記載の脳損傷治療用細胞構造体。
- 請求項1から15の何れか一項に記載の脳損傷治療用細胞構造体を複数個融合することにより得られる、脳損傷治療用細胞構造体。
- タップ密度が10mg/cm3以上500mg/cm3以下であるか、又は二次元断面像における断面積の平方根÷周囲長の値が0.01以上0.13以下である生体親和性高分子ブロックと細胞とを混合し、10時間以上培養する工程を含む、請求項1から16の何れか一項に記載の脳損傷治療用細胞構造体の製造方法。
- 請求項1から16の何れか一項に記載の脳損傷治療用細胞構造体を含む、脳損傷治療剤。
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EP3156081A1 (en) | 2017-04-19 |
US20170095595A1 (en) | 2017-04-06 |
US10500311B2 (en) | 2019-12-10 |
JPWO2015194494A1 (ja) | 2017-04-20 |
JP6330042B2 (ja) | 2018-05-23 |
EP3156081A4 (en) | 2017-08-16 |
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