JP2018174929A - Culture fluid additives for mammalian cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To proliferate undifferentiated cells in a cell culture medium in a cell culture medium in order to maintain undifferentiated cells in an undifferentiated state or to differentiate / proliferate into target differentiated cells or tissues. To provide a new means for slowly releasing a factor or various humoral factors that affect the differentiation and proliferation of cells. SOLUTION: A humoral factor that proliferates undifferentiated mammalian cells in an undifferentiated state on a substrate having a porous structure having at least a hydrophilic surface, or an influence on the differentiation and proliferation of mammalian cells. A cell culture aid to which a humoral factor is adsorbed, or a cell culture aid having a calcium phosphate layer containing these humoral factors on the surface of a substrate having at least a hydrophilic surface. By putting this into a cell culture medium, these humoral factors can be slowly released into the medium for a long period of time. [Selection diagram] Fig. 4

Description

  The present invention relates to the field of cell culture technology, and further to the field of regenerative medicine and medical fields utilizing the same, and in particular, maintenance culture of undifferentiated cells such as mammalian stem cells and progenitor cells in an undifferentiated state The present invention also relates to a cell culture supplement for differentiating and proliferating target cells into target cells.

In recent years, the patient's loss was caused by differentiating in the intended direction using undifferentiated cells such as mammalian embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or precursor cells. Reproduce various tissues and organs in vivo and in vitro, or artificially prepare various tissues and organs of mammals, and use them to screen new medicines in a situation closer to the actual living environment. The research and development of technologies related to regenerative medicine and drug discovery, such as, are being promoted in various fields.
In using these undifferentiated cells, it is important to maintain the cells before use in an undifferentiated state.
In addition, in order to differentiate these undifferentiated cells, it is important to appropriately differentiate / proliferate target differentiated cells and tissues.

The above-mentioned stem cells and progenitor cells are, for example, fibroblast growth factor-2 (also referred to as basic fibroblast growth factor, FGF-β, FGF-2, bFGF, etc. in the present specification, hereinafter referred to as bFGF) in a medium. It is known that the addition of a fluid factor can maintain the undifferentiated state.
For example, Patent Document 1 discloses that embryonic stem cells are cultured in a medium free of feeder cells or serum and under feeder-free / serum-free culture conditions in a medium to which bFGF is added at a concentration of about 10 ng / mL. Maintaining in a differentiated state is disclosed. However, the half life of bFGF in the culture medium is short, and in order to keep cells in an undifferentiated state for a necessary period in this method, bFGF needs to be frequently replenished in the culture medium.
In addition, it is known that various liquid factors such as epidermal growth factor and vascular endothelial growth factor affect the differentiation and proliferation of undifferentiated cells, and the above-mentioned undifferentiated stem cells and progenitor cells In order to differentiate and proliferate cells into target differentiated cells and tissues by culture, it is necessary to appropriately add these humoral factors, but many of these humoral factors also have half-lives in the medium. It is known to be short.

As a method of solving such a defect, a method of adding a growth factor in the form of a sustained release composition which continuously releases in the medium has been developed (patent document 2, non-patent document 1). This method uses a biodegradable polyester poly (DL-lactide coglycolide) (PLGA) in which microbeads of bFGF are supported as a sustained release composition.
By this method, the frequency of bFGF supplementation can be reduced by releasing bFGF into the medium slowly.

  Also, although it is not a technique for releasing the drug into the culture medium, as a method for releasing the drug such as a growth factor into the environment such as body fluid, heparin-conjugated growth factor is used to the substrate on which heparin is covalently crosslinked. Are bound and used as a heparin-binding growth factor sustained release agent (Patent Document 3), using a drug inclusion carrier consisting of fine particles having a mean particle size of less than 10 μm, composed of low molecular weight heparin and protamine, It is known that a drug such as bFGF is included and slowly released into the environment (Patent Document 4).

Japanese Patent Application Publication No. 2009-542247 Japanese Patent Application Publication No. 2013-529932 JP 2001-233786 A JP, 2011-026202, A

Steven Lotz et al. Plos one, Volume 8, e56289, 2013

The method of using bFGF supported in PLGA microbeads as a sustained release composition as described above is that PLGA elutes bFGF into the medium as it is gradually degraded in the medium. Dissolving and mixing of lactic acid and the like generated in the medium into the medium may cause the medium to gradually deteriorate and the pH of the culture solution to decrease, or the decomposition product may be pulverized and be taken into cultured cells, etc. There is. Moreover, since this PLGA bead sinks to the bottom of the culture dish and contacts the cells, the possibility of affecting the cells by physical stimulation to the cells, interaction with the cell surface, etc. can not be denied.
In the present invention, a humoral factor that allows undifferentiated cells such as bFGF to proliferate in an undifferentiated state in the medium, or various humoral factors that affect cell differentiation / proliferation are sustained-released, whereby stem cells etc. To provide a more effective method for maintaining undifferentiated cells in an undifferentiated state or differentiating / proliferating undifferentiated cells into target differentiated cells or tissues, without the above disadvantages of the prior art. As an issue.

The present inventors hydrophilize the surface of the porous substrate, and immerse the surface in a bFGF-containing solution so that bFGF can be adsorbed onto the porous substrate, and the porous substrate is introduced into the medium. By doing this, it was found that bFGF was well released in the medium.
Specifically, the present inventors hydrophilize the surface by plasma treating a commercially available polyethylene non-woven fabric and a commercially available polypropylene membrane filter, and immersing the surface in a bFGF-containing solution. BFGF is well adsorbed to these non-woven fabrics etc., and when it is put into a medium for cell culture (culture medium), it is found that bFGF is sustainedly released well, and the non-woven fabrics etc. It was found that it floated on its top surface without sinking.
Thus, by adjusting the porosity of the porous substrate appropriately, bFGF can be adsorbed well and sustained release can be performed well.
Moreover, the base material can be floated on the surface of the culture medium by appropriately selecting the material of the porous base material and setting the specific gravity of the base material smaller than the specific gravity of the culture medium. In this way, it is possible to easily load and remove the substrate from the medium, and to avoid direct contact of cells cultured in the medium with the substrate.
The form of the porous substrate is not particularly limited, and may be in the form of beads or the like, but if it is formed into a planar form such as a flat plate or a sheet like the non-woven fabric and membrane filter described above, the substrate is added to the culture medium・ It becomes easier to remove.

  The present inventors further immerse the base material having the surface subjected to hydrophilization treatment in a solution for calcium phosphate precipitation to which bFGF is added, and the base material obtained by depositing the bFGF-calcium phosphate composite layer on the base material surface Also, by slowly releasing bFGF well into the medium, and also in this case, by appropriately selecting the material of the base, the base is made to be a medium by making the specific gravity of the base smaller than the specific gravity of the medium. It has been found that the substrate itself may not necessarily have a porous structure, because it can float on the surface, and in this case, bFGF is slowly released from the calcium phosphate composite layer.

The present inventors further adsorb heparin with bFGF to a porous substrate, or deposit a bFGF / heparin-calcium phosphate composite layer on the substrate surface using a solution for calcium phosphate precipitation with heparin added with bFGF Thus, it was found that bFGF was more favorably sustained-released from the obtained substrates into the medium.
The present invention has been made based on these findings obtained by the present inventors.

That is, this application provides the following invention.
(1) A liquid factor that causes undifferentiated cells of a mammal to proliferate undifferentiated in a base material having a porous structure at least the surface of which is hydrophilic, or a liquid that affects differentiation and growth of mammalian cells Cell culture supplements that are adsorbed by sex factors.
<2> The substrate having a porous structure is a fiber assembly, a porous membrane, a substrate having a porous surface only, or a substrate having the porous substrate adhered to the surface, <1 Cell culture supplement as described in>.
The cell culture adjuvant as described in <1> or <2> in which the base material which has <3> porous structure contains polyethylene, a polypropylene, or a polystyrene as a raw material.
<4> The cell culture support according to any one of <1> to <3>, which has a specific gravity smaller than that of the cell culture solution.
<5> The cell culture according to any one of <1> to <4>, wherein the liquid factor is a factor capable of binding to heparin, and a sulfated polysaccharide is further adsorbed on a substrate having a porous structure. Adjuvant.
<6> The cell culture support according to any one of <1> to <5>, wherein the humoral factor is fibroblast growth factor-2 (bFGF).
(7) A liquid factor that hydrophilizes the surface of a substrate having a porous structure and causes undifferentiated cells of a mammal to grow undifferentiated as such, or a liquid that affects differentiation and growth of mammalian cells The cell culture according to any one of <1> to <4> and <6>, wherein the factor is adsorbed to a substrate having a porous structure by immersing in a solution containing the factor. Method of producing adjuvants.
<8> A liquid factor that hydrophilizes the surface of a substrate having a porous structure and causes undifferentiated cells of a mammal to grow undifferentiated as such, or a liquid that affects differentiation and growth of mammalian cells The factor and the sulfated polysaccharide are adsorbed to a substrate having a porous structure by being immersed in a solution containing the factor and the sulfated polysaccharide, described in <5> or <6> Method for producing cell culture supplements.
<9> The method for producing a cell culture support according to <7> or <8>, wherein the surface of the substrate having a porous structure is hydrophilized by plasma treatment or acid treatment.
The cell culture supplement described in any one of <10><1> to <6> is added to a cell culture medium for culturing a mammalian cell, and the mammal is released while releasing the liquid factor into the medium. How to culture cells.
<11> A mammalian cell is an undifferentiated cell, a humoral factor is a humoral factor that causes an undifferentiated cell to grow in an undifferentiated state, and the undifferentiated cell is maintained in an undifferentiated state and cultured. The method according to <10>, characterized in that
<12> The humoral factor is a humoral factor that causes mammalian cells to differentiate and proliferate mammalian cells into differentiated cells or tissues, and is characterized in that mammalian cells are differentiated and proliferated into differentiated cells or tissues by culture, <10> The method described in.
<13> A humoral factor that causes an undifferentiated cell of a mammal to proliferate undifferentiated on the surface of a substrate at least the surface of which has hydrophilicity, or a humoral factor that affects differentiation and proliferation of mammalian cells A cell culture supplement having a calcium phosphate layer containing.
<14> A substrate having a porous structure in which the substrate is a fiber assembly, a porous membrane, a substrate having a porous surface only, or a substrate having the porous substrate adhered to the surface. The cell culture adjuvant as described in <13> which is.
<15> The cell culture support according to <13> or <14>, wherein the substrate contains polyethylene, polypropylene or polystyrene as a material.
<16> The cell culture supplement according to any one of <13> to <15>, which has a specific gravity smaller than that of the cell culture solution.
<17> The cell culture support according to any one of <13> to <16>, wherein the humoral factor is a factor capable of binding to heparin, and the calcium phosphate layer further contains a sulfated polysaccharide.
<18> The cell culture support according to any one of <13> to <17>, wherein the humoral factor is fibroblast growth factor-2 (bFGF).
<19> Calcium phosphate containing a humoral factor that hydrophilizes the surface of a substrate and allows undifferentiated cells of a mammal to proliferate undifferentiated, or a humoral factor that affects differentiation and proliferation of mammalian cells Production of the cell culture adjuvant according to any one of <13> to <16> and <18>, characterized in that a calcium phosphate layer containing the factor is formed on the substrate surface by immersion in a supersaturated solution. Method.
<20> A humoral factor that hydrophilizes the surface of a substrate and allows undifferentiated cells of a mammal to grow undifferentiated, or a humoral factor that affects differentiation and proliferation of mammalian cells, and sulfate The cell culture adjuvant according to <17> or <18>, characterized in that the calcium phosphate layer containing the factor and the sulfated polysaccharide is formed on the substrate surface by immersing in a calcium phosphate supersaturated solution containing the immobilized polysaccharide. Manufacturing method.
<21> The method for producing a cell culture supplement according to <19> or <20>, wherein the surface of the substrate is hydrophilized by plasma treatment or acid treatment.
The cell culture supplement as described in any one of <22><13> to <18> is added to a cell culture medium for culturing a mammalian cell, and the mammal is released while releasing the liquid factor into the medium. How to culture cells.
<23> A mammalian cell is an undifferentiated cell, a humoral factor is a humoral factor that causes an undifferentiated cell to proliferate in an undifferentiated state, and the undifferentiated cell is maintained in an undifferentiated state and cultured. The method according to <22>, characterized in that
<24> The humoral factor is a humoral factor that causes mammalian cells to differentiate and proliferate mammalian cells into differentiated cells or tissues, and is characterized in that mammalian cells are differentiated and proliferated into differentiated cells or tissues by culturing, <22> The method described in.

According to the present invention, a readily available base material having a porous structure such as a commercially available non-woven fabric made of polyethylene or polypropylene or a membrane filter can be used as the base material, and the surface is made hydrophilic by subjecting it to a hydrophilization treatment. These formed porous substrates are immersed in a solution containing a liquid factor such as bFGF to adsorb the liquid factor or the surface of the calcium phosphate precipitation solution to which the liquid factor is added. It is possible to obtain a controlled release agent for releasing the liquid factor into the medium by a simple and inexpensive process of precipitating the liquid factor-calcium phosphate composite layer. In the case of depositing the liquid factor-calcium phosphate composite layer on the surface, the base material itself does not necessarily have a porous structure, since the liquid factor is released slowly from the calcium phosphate composite layer. From the viewpoint of increasing the formation amount, it is preferable to have a shape having a high specific surface area such as a porous structure or fine particles.
In the present invention, by appropriately selecting the material of the base material, by setting the specific gravity of the base material to be smaller than the specific gravity of the culture medium, it is possible to obtain a sustained release agent floating on the upper surface of the culture solution. . This facilitates the addition and removal of the sustained release agent to the culture medium, and prevents the sustained release agent from directly contacting the cultured cells.
In the present invention, it is possible to use a chemically stable material as a substrate itself, and when the sustained release agent is introduced into the medium, decomposition products of the substrate are eluted in the medium. , There is no risk of affecting cultured cells.

SEM image of PS (polystyrene) flat substrate, PE (polyethylene) non-woven fabric (thin), PE non-woven fabric (thick), PP (polypropylene) filter (pores), PP filter (large pore) (each left: low magnification image , Each right: high magnification image). SEM image after plasma treatment of PE non-woven fabric (thin), PE non-woven fabric (thick), PP filter (pores), PP filter (large pore). Photograph (upper) of PE non-woven fabric (thickness) and PP filter (pore) floated in culture solution, and schematic view seen from the side (lower). Graph showing bFGF elution behavior to a culture solution of each base material immersed for 30 minutes (upper) or 24 hours (lower) in bFGF adsorption solution after plasma treatment (■ broken line: PE non-woven fabric (thin), ◆ broken line : PE non-woven fabric (thickness), ● solid line: PP filter (pore), 実 線 solid line: PP filter (large pore), × dotted line: PS flat plate-like substrate). The SEM image (left) of the substrate surface plasma-treated in various conditions, and the graph which shows the contact angle of ultrapure water (right). The graph which shows the bFGF residual amount in each solution for bFGF adsorption after adsorbing-processing the base material plasma-processed on various conditions. Graph showing bFGF elution behavior to a culture solution of each substrate immersed in the bFGF adsorption solution for 24 hours after plasma treatment under various conditions (× dotted line: untreated, ● solid line: 0.05 W / cm ² , ▲ Broken line: 0.1 W / cm ² , ◆ broken line: 0.2 W / cm ² , ■ Solid line: 0.5 W / cm ² ). The graph which shows the bFGF residual amount in each solution for bFGF adsorption after carrying out the adsorption process to the solution for bFGF adsorption for the various time for the base material by which the plasma process was carried out. During adsorption treatment, when the bFGF adsorption solution is allowed to stand (top) and shaken (bottom) (● dashed line: no immersion of base material, ■ solid line: immersion of base material) Graph showing the amount of bFGF remaining in each bFGF adsorption solution after immersing the plasma-treated substrate in various concentrations of bFGF adsorption solution while shaking for 24 hours (dot pattern: no immersion of substrate) , Gray pattern: with immersion of the substrate). Graph showing bFGF elution behavior to culture solution of each base material immersed in various concentrations of bFGF adsorption solution after plasma treatment for 24 hours (● solid line: 12 μg / mL, ● broken line: 8 μg / mL, ■ solid line : 4 μg / mL, ■: broken line: 2 μg / mL,: 1 solid line: 1 μg / mL, 破 線 broken line: 0.5 μg / mL, ◆ solid line: 0.2 μg / mL, ◆ broken line: 0.1 μg / mL, × solid line: 0 μg / mL) The SEM image of each base material immersed in the calcium phosphate supersaturated solution which added bFGF after plasma treatment for 24 hours. Graph showing bFGF elution behavior to a culture solution of PS and PP base material soaked in calcium phosphate supersaturated solution added with bFGF for 24 hours after plasma treatment (left) (● solid line: PP filter (pores), 実 線 solid line : PP filter (large pore), □ solid line: PP net filter, × dotted line: PS flat plate-like substrate, and SEM image of substrate before plasma treatment (right). Graph showing bFGF elution behavior to culture solution of each base material immersed in calcium phosphate supersaturated solution added with bFGF for 24 hours after plasma treatment (■ broken line: PE non-woven (thin), ◆ broken line: PE non-woven (thick) , ● Solid line: PP filter (pores), × dotted line: PS flat substrate). The SEM image of each base material immersed in the calcium phosphate supersaturated solution which added various bFGF after plasma treatment for 24 hours. Graph showing bFGF elution behavior to a culture solution of each substrate immersed in calcium phosphate supersaturated solution added with various bFGF after plasma treatment (■ solid line: thick-low temperature-F4,-solid line: high-low temperature -F10, 実 線 solid line: thick-high temperature-F10, ◆ solid line: thin-low temperature-F10). SEM image of the surface of PE mesh sheet and PE porous sheet. SEM image of each substrate (without plasma treatment) immersed in calcium phosphate supersaturated solution with bFGF added for 24 hours. Graph showing bFGF elution behavior to culture solution (upper: no plasma treatment, lower: with plasma treatment) of each substrate immersed in calcium phosphate supersaturated solution to which bFGF is added for 24 hours (solid line ●: PE (mesh), ■ Solid line: PE (porous), × dotted line: PS flat substrate). SEM images of PVDF and ester substrate surfaces. Graph showing bFGF elution behavior to a culture solution of PVDF base material soaked for 6 hours in bFGF and heparin adsorption solution (upper: bFGF 0.1 μg / mL, lower: bFGF 1.0 μg / mL) (heparin in adsorbed liquid) Concentration: 破 線 broken line: 0 unit / mL, 実 線 solid line: 0.1 unit / mL, solid line ●: 1 unit / mL, ■ solid line: 10 unit / mL). Graph showing bFGF elution behavior to a culture solution (upper: bFGF 0.1 μg / mL, lower: bFGF 1.0 μg / mL) of the ester base material immersed in the bFGF and heparin adsorption solution for 6 hours (heparin in the adsorption solution) Concentration: 破 線 broken line: 0 unit / mL, 実 線 solid line: 0.1 unit / mL, solid line ●: 1 unit / mL, ■ solid line: 10 unit / mL). Graph showing bFGF elution behavior to a culture solution of PS plate-like substrate soaked in calcium phosphate supersaturated solution added with bFGF and heparin for 24 hours (heparin concentration in supersaturated solution; ◆ solid line: 0 unit / mL, solid line: 0.1 unit / mL, solid line ●: 3.6 unit / mL). SEM image of beaded PS substrate soaked in calcium phosphate supersaturated solution for 24 hours. Condition (1) (bFGF sustained-release porous substrate (bFGF adsorption type) installed, culture fluid not exchanged), condition (2) (bFGF-added culture fluid is replaced daily), condition (3) (bFGF non-added culture fluid) The culture medium of human iPS cell culture day 4 (dot pattern) and day 8 (gray pattern) in daily change), and the condition (4) (culture solution without seeding human iPS cells in condition (1)) Graph showing bFGF concentrations in cultures on day 4 (dot pattern) and day 8 (grey pattern). Phase contrast microscopic images of human iPS cells on the first, fourth, fifth and eighth days of culture under the conditions (1) to (3). The fluorescence microscope image of the human iPS cell stained by each method after culture | cultivation on condition (1) or (2). The graph which shows the M-CSF elution behavior to the culture solution of the base material which was immersed in the solution (4 micrograms / mL) for M-CSF adsorption for 24 hours after plasma treatment. The graph which shows the TNFα elution behavior to the culture solution of the base material which was immersed in the solution (4 micrograms / mL) for TNFα adsorption after plasma treatment for 24 hours.

According to the present invention, it is possible to maintain and culture undifferentiated cells in an undifferentiated state by slowly releasing a humoral factor that causes undifferentiated mammalian cells to proliferate in an undifferentiated state into the cell culture medium. . The humoral factor in the present invention is a hormone, cytokine, neurotransmitter, etc., a signal transduction substance that affects the undifferentiated maintenance / differentiation / proliferation of mammalian cells by transmitting a signal to the cells, and is usually used in a medium. It has the property of dissolving. Depending on the role of humoral factors, in addition to undifferentiated maintenance factors, they can be classified into growth factors, differentiation inducers, adhesion factors, transcription factors, and the like.
As the undifferentiated maintenance factor used in the present invention, fibroblast growth factor (FGF) family, leukemia inhibitory factor (LIF), CCL2, transforming growth factor-beta (TGF-beta) family, insulin, insulin-like growth factor ( And IGF) family, transferrin family and the like. Among them, fibroblast growth factor-2 (also referred to as basic fibroblast growth factor, FGF-β, FGF-2, bFGF, etc.) is preferable.

According to the present invention, mammalian cells are differentiated and proliferated into differentiated cells or tissues by culturing various humoral factors that affect differentiation and proliferation of mammalian cells in a cell culture medium with slow release. be able to.
In this way, the object of producing the target cell from undifferentiated cells, efficiently culturing the target cell, and analyzing the interaction between the cell and the liquid factor is achieved.
The liquid factor that affects cell growth, differentiation, etc. is not particularly limited as long as it is a liquid factor that dissolves in a culture medium. For example, epidermal growth factor (EGF) family, connective tissue growth factor family, vascular endothelial growth factor (VEGF) family, neurotrophic factor family, glial cell line-derived neurotrophic factor (GDNF) family, platelet derived growth factor (PDGF) ) Family, hepatocyte growth factor (HGF) family, bone morphogenetic protein (BMP) family, transforming growth factor-α (TGF-α) family, transforming growth factor-β (TGF-β) family, activin family, human Growth hormone, heregulin family, insulin-like growth factor (IGF) family, interleukin (IL) family, interferon (INF) family, granulocyte colony stimulating factor (G-CSF) family, granulocyte macrophage colony stimulating factor (GM-) CSF) family, macrophage colony stimulating factor (M-CSF) family Activated protein (MAP) kinase family, Flt3 ligand, stem cell factor (SCF) family, tumor necrosis factor (TNF) family, gene transcription regulatory factor (GATA) family, erythropoietin, thrombopoietin, etc., but not limited thereto .

  Examples of the base material used in the present invention include polyethylene, polystyrene, polypropylene, ABS resin, AS resin, polyvinyl chloride, acrylic resin, polyvinyl alcohol, polyvinyl pyrrolidone, nylon, cellulose, acetal resin, polycarbonate, polyethylene terephthalate, Polybutylene terephthalate, polyvinylidene chloride, polyvinylidene fluoride, polysulfone, polyurethane, polyimide resin, polyetherimide, polyamide imide, acetal resin, phenol resin, melamine resin, epoxy resin, copolymers and composites of these, etc. A base material is mentioned.

These base materials are, for example, acid treatment, alkali treatment, oxidation treatment, plasma treatment, corona discharge treatment, flame treatment, glow discharge treatment, ion sputtering treatment, laser irradiation, ultraviolet irradiation, gamma ray irradiation, surface polishing, etching treatment, Introduction of hydrophilic functional groups (by silane coupling treatment, graft polymerization of molecules with hydrophilic functional groups, etc.), surface coating of hydrophilic film / coating material, surface coating of surfactant etc. Can be In addition, the base material surface can be made hydrophilic by mixing and kneading a hydrophilic polymer or an inorganic filler.
When the material itself exhibits sufficient hydrophilicity for permeation and contact of a culture medium, such as certain polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyvinyl pyrrolidone, polysulfone, etc., a hydrophilization treatment is performed. It does not have to be.

When the substrate of the present invention has a porous structure, the shape of the substrate is preferably an aggregate of fibers such as non-woven fabric, woven fabric and knitted fabric, or a porous membrane or flat plate such as a membrane filter or mesh sheet. Any of these porous substrates can be produced by known production methods.
In the present invention, a substrate having a porous structure refers to a substrate having a surface roughness larger than that of a substrate having a smooth surface and a large surface area (100 mm ² or more for a 10 mm square substrate). . The surface roughness is preferably 1 to 90 μm, more preferably 2 to 10 μm in height arithmetic average (Sa) in the height direction. The surface area (A) of one side (theoretical surface area in the case of perfect smoothness = 100 mm ² ) of a substrate of 10 mm × 10 mm is preferably 150 to 2500 mm ² (1.5 to 25 times the perfect smooth surface), more preferably It is 150-500 mm < ² > (1.5-5 times the perfect smooth surface).
However, in the case of a substrate having a large opening (a gap between fibers and fibers) in the mesh sheet, even if the effect of increasing the surface roughness and surface area of the entire substrate is large, the adsorption liquid is actually adsorbed when suspended in the culture medium. And, when the effective surface area that can be in contact with the culture medium is small, it is necessary to be careful because the desired sustained release performance can not be achieved.
It is desirable that the porous structure to be provided to the substrate has a large effective surface area capable of being in contact with the adsorption solution and the culture medium while suspended in the culture medium so as to ensure a sufficient contact area with the adsorption solution and the culture medium. . As such a porous structure, it is desirable that it is a porous structure of micrometer scale, and it is desirable that it is a connected porous structure in which the pores and the pores are continuously connected, but it is not limited thereto. Examples of preferred porous substrate, Sa is 5 to 6 .mu.m, A polyethylene nonwoven fabric or 160~170mm ^2, Sa is 2 [mu] m, A is like polypropylene filter 240 mm ^2.
In addition, the base material does not have to be porous throughout, and at least one side of the base material may have a porous structure. A substrate having a porous structure on at least one side of the substrate can also be obtained by combining a plurality of substrates, such as laminating a porous substrate and a dense substrate. Alternatively, acid, alkali, oxidation, plasma treatment, corona discharge treatment, flame treatment, glow discharge treatment, ion sputtering treatment, laser irradiation, ultraviolet irradiation, gamma irradiation, surface polishing, etching treatment to a dense substrate The surface may be made porous by applying a surface coating of a porous film or a coating material.

In order to suspend the substrate of the present invention in a culture medium, the specific gravity of the substrate is preferably less than 1. As such a low specific gravity substrate, polyethylene, polystyrene, polypropylene, ABS resin and the like can be mentioned.
By combining the base material having a specific gravity of 1 or more with the base material having a specific gravity of less than 1, the specific gravity of the whole base material may be less than 1 and the culture medium floatability may be secured. Alternatively, air bubbles may be introduced into the base material or a low specific gravity component may be mixed to make the specific gravity of the whole base material less than 1, thereby securing the medium floating property.

As a calcium phosphate supersaturated solution to be used in the present invention, an aqueous solution containing at least a calcium ion and a phosphate ion at or above a saturation concentration is used. However, the solution may be a solution which is unsaturated with respect to calcium phosphate immediately after preparation of the solution, and the component concentration changes with time to be supersaturated. Alternatively, it may be converted to a supersaturated solution by adding operations such as raising the temperature of the solution, adding an alkalizing agent, adjusting the ionic strength, etc. at the time of use.
The calcium phosphate supersaturated solution further contains components for adjusting the ionic strength of the solution (such as NaCl), components for adjusting the pH (TRIS buffer, HEPES buffer, etc.), and components for controlling nucleation and growth of calcium phosphate ( Magnesium, carbonate ion, etc. may be contained.
As such a calcium phosphate supersaturated solution, for example, a simulated body fluid having an inorganic ion concentration substantially equal to that of human body fluid, a solution (such as 5-fold concentration simulated body fluid) in which a specific ion concentration of the simulated body fluid is increased, CP solution (Uchida et al. Adv. Mater. Volume 16, 1071, 2004), RKM solution (Sogo et al. Curr. Appl. Phys. Volume 5, 526, 2005), Hanks solution etc. can be used. Any of these supersaturated solutions can be prepared by known methods. The pH of the calcium phosphate supersaturated solution may be neutral, preferably 5 to 12, preferably 6 to 10, and more preferably 7 to 8.

The sulfated polysaccharides used in the present invention include heparin, heparan sulfate, heparin-derived oligosaccharides, heparan sulfate-derived oligosaccharides and the like. Among them, heparin and heparan sulfate are preferable.
When the liquid factor is a heparin-binding protein, binding of these sulfated polysaccharides enables the liquid factor to be used in the adsorption liquid or calcium phosphate supersaturated solution used for producing the sustained release agent of the present invention. And in the medium after sustained release.

  EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1 Examination of Substrate in Adsorption Method After a porous substrate was subjected to a hydrophilization treatment, fibroblast growth factor-2 (bFGF) was adsorbed. By using a porous substrate having a large surface area, it was confirmed that the elution amount of bFGF into the culture solution is increased as compared to a polystyrene (PS) substrate having a dense and smooth surface.

Preparation of bFGF sustained-release porous substrate (bFGF adsorption type) Using the following four porous substrates (Figure 1) made of polyethylene (PE) and polypropylene (PP), bFGF was adsorbed according to the following procedure The Also, for comparison, a PS flat substrate having a dense and smooth surface was used. In addition, all base materials other than PS base material float in water.

・ PE non-woven fabric (thin): Tyvek 1442R (Asahi, DuPont Flash Span Products) thickness about 145μm
・ PE non-woven fabric (thick): Bexar S (Tyvek) (made by Asahi DuPont Flash Spun Products) Thickness about 160μm
・ PP filter (pore): AN 1204700 (Merck Millipore) pore size 1.2 μm, thickness 150 μm
・ PP filter (large pore): AN1H04700 (made by Merck Millipore) pore size 10 μm, thickness 150 μm

First, each porous substrate was cut into pieces of 10 mm × 10 mm in size with a cutter. The PE non-woven fabric was ultrasonically cleaned in ultrapure water for 30 minutes and then ultrasonically cleaned in ethanol for 30 minutes. The PP filter was used as it was without washing. A PS flat substrate is prepared by melting and molding PS pellets (manufactured by Aldrich Chemical) into a 1 mm thick substrate cut into 10 mm × 10 mm pieces, and the surface is an abrasive (Tamiya compound (finished grain)) And then ultrasonically cleaned with ethanol.
The surface roughness (amplitude arithmetic mean in the height direction: Sa) of each substrate was measured by a 3D measurement laser microscope (LEXT OLS 4100, manufactured by Olympus). Further, the surface area (A) per one side of the substrate (size: 10 mm × 10 mm = 100 mm ² ) was calculated. The results are shown below (numbers are the mean of three measurements ± standard deviation).

PS flat substrate Sa = 0.5 ± 0.0 μm, A = 109.3 ± 1.1 mm ²
PE non-woven fabric (thin) Sa = 5.3 ± 0.6 μm, A = 171 ± 12 mm ²
PE non-woven fabric (thickness) Sa = 5.6 ± 1.1 μm, A = 157 ± 4 mm ²
PP filter (pore) Sa = 2.4 ± 0.3 μm, A = 237 ± 17 mm ²
PP filter (large hole) Sa = 4.8 ± 0.0 μm, A = 419 ± 6 mm ²

From this result, it was confirmed that all porous substrates have large surface roughness (5 to 11 times) and surface area (1.5 to 4 times) as compared to PS flat substrates.
Subsequently, each substrate was subjected to oxygen plasma treatment (30 Pa, 13.56 MHz, 30 seconds) for hydrophilization using a compact etcher FA-1 (manufactured by Samco Inc.). Power density of the plasma treatment, PE nonwoven (thin) and PE nonwoven 0.1 W / cm ² for (thick), PP filter for the (large hole) 0.3W / cm ^2, PP filter (pore) And 0.5 W / cm ² for PS and flat plate substrates. The above processing conditions were determined from the results of preliminary examination as conditions capable of hydrophilizing the surface of the substrate without deforming or discoloring the substrate. After plasma treatment, each substrate was sterilized with ethylene oxide (EOG) gas.
By adding bFGF (1 mg / mL, manufactured by Katayama Chemical Industry Co., Ltd.) to a final concentration of 4 μg / mL in phosphate buffered saline (D-PBS (-), manufactured by Wako Pure Chemical Industries, Ltd., hereinafter PBS), An adsorption solution was prepared. 2 mL of the adsorption solution was dispensed into the wells of a 24-well plate, and each substrate was immersed in the adsorption solution at 25 ° C. for 30 minutes or 24 hours. At this time, since the porous substrate floats on water, the plasma-treated surface was placed with the treated surface down so that the plasma-treated surface was in contact with the adsorption solution. Since the PS flat plate-like substrate was submerged in water, it was placed with the plasma-treated surface on top. The 24-well plate was placed in a zippered plastic bag (Unipack) to prevent evaporation of the adsorption solution, and was allowed to stand in a cool incubator maintained at 25 ° C. After immersion, the substrate was removed from the solution and washed three times (1 second) with 2 mL PBS (without bFGF added) for 3 times.

Evaluation of Substrates FIG. 2 shows a scanning electron microscope (SEM) image of each porous substrate after plasma treatment. On the surface of the PP filter (pores), formation of a submicron-scale dot-like concavo-convex structure was observed, but for the other substrates, a remarkable structural change was observed compared to before the plasma treatment (FIG. 1) I was not able to admit. When ultrapure water was dropped on the substrate surface before and after the plasma treatment, it was found that the water wettability of the substrate surface was improved by the plasma treatment for any substrate. It is considered that the hydrophobic PP and PE surfaces were hydrophilized by plasma treatment.

bFGF elution behavior 2 mL of a culture solution (Essential 6 Medium, manufactured by Thermo Fisher Scientific) used for culturing pluripotent stem cells is dispensed into the wells of a 24-well plate, and each substrate on which bFGF is adsorbed according to the above procedure Were placed so that the treated surface (bFGF adsorption surface) was in contact with the culture solution (porous substrate: treated surface is lower, PS flat substrate: treated surface is upper). FIG. 3 shows photographs (top) and schematic views (bottom) of PE non-woven fabric (thickness) and PP filter (pores) floated in a culture solution. After placing each substrate, the 24-well plate was placed in a 37 ° C. carbonic acid incubator. After 1, 2 and 3 days, 150 μL of the culture solution (bFGF eluate) in the wells was collected, and 150 μL of a new culture solution was added. The collected culture fluid (bFGF eluate) was immediately stored at -80 ° C and thawed at the time of measuring bFGF concentration. The bFGF concentration in the eluate was measured using an ELISA kit (Quatikine ELISA Human FGF basic Immunoassay, manufactured by R & D). The calibration curve was prepared using bFGF (manufactured by Katayama Chemical Industry Co., Ltd.).
The bFGF elution behavior from each substrate is shown in FIG. The amount of bFGF elution increased in the substrate for 24 hours (FIG. 4 lower) than for the substrate with an adsorption time of 30 minutes (FIG. 4 upper). It is considered that by increasing the adsorption time, a larger amount of bFGF was supported on the substrate. From the result of the adsorption time of 24 hours (FIG. 4 lower), the bFGF elution amount can be increased by using a porous substrate having a large surface roughness and a large surface area with respect to a PS flat substrate having a dense and smooth surface Was confirmed. In the PP filter, the bFGF elution amount was larger in the filter with the smaller pore size than in the large pore size. The PE non-woven fabric (thin) and the PP filter (pore) achieved a bFGF concentration of 10 ng / mL or more after 1 day, and kept 8 ng / mL after 3 days.
When bFGF (Katayama Chemical Industries) was added to the same culture solution at an initial concentration of 10 ng / mL, bFGF detected by ELISA after 1 day was 0.5 ng / mL or less. That is, bFGF in a free state changes to a state in which 95% or more can not be detected by ELISA within one day in a culture solution environment. This is because the half life of bFGF is extremely short.
The bFGF sustained-release porous substrate (bFGF adsorption type) prepared by the present adsorption method floated in the culture solution (without contacting the cells) and maintained a bFGF concentration of 8 to 10 ng / mL for 3 days. From the above, it can be said that the present base material satisfies the cell non-contactability desired for pluripotent maintenance culture of pluripotent stem cells and the bFGF long-term sustained release performance (Non-patent document 1). Moreover, this base material can be prepared without using drugs such as animal-derived heparin, synthetic polymers, organic solvents and the like used in prior products (Non-Patent Document 1), and bFGF long-term sustained release equivalent to prior products Performance has been achieved.

(Example 2) Examination of treatment conditions (hydrophilization treatment conditions, adsorption treatment time, effect of shaking, bFGF concentration) in the adsorption method As a result of subjecting the porous substrate to hydrophilization treatment under various conditions, hydrophilization treatment It was confirmed that the amount of bFGF adsorbed to the substrate and the amount eluted into the culture solution increased. As a result of performing adsorption treatment for various times, the amount of bFGF adsorbed to the substrate increased with the passage of time, and reached equilibrium in about 24 hours. A comparison between the case in which the bFGF adsorption solution was allowed to stand and the case in which the solution was shaken during the adsorption treatment confirmed that shaking could efficiently adsorb bFGF. In addition, as a result of performing adsorption treatment using solutions for bFGF adsorption at various concentrations, as the bFGF concentration of the solution for adsorption increases, the amount of bFGF adsorbed on the substrate and the elution amount into the culture solution increase. It was confirmed.

Preparation of bFGF sustained-release porous substrate (bFGF adsorption type) (examination of hydrophilization treatment conditions)
PE non-woven fabric equivalent to PE non-woven fabric confirmed to have good bFGF sustained-release performance in Example 1 and used for packaging of medical devices etc. (Asahi / Dupont flush span products Tyvek 1073 B, thickness approx. 178 μm) was prepared. The substrate was cut into pieces of 10 mm × 10 mm in size, subjected to ultrasonic cleaning in ultrapure water for 30 minutes, and then ultrasonically washed in ethanol for 30 minutes to form a substrate.
Oxygen plasma treatment (30 Pa, 13.56 MHz, 30 seconds) with various power densities (0.05, 0.1, 0.2, 0.5 W / cm ² ) using a compact etcher FA-1 (manufactured by Samco Inc.) for each base material Applied. The substrate after plasma treatment was sterilized with EOG gas, immersed in PBS (adsorption solution) containing bFGF (4 μg / mL) at 25 ° C. for 24 hours in the same manner as Example 1, and washed. However, in this example, 1 mL of the adsorption solution (2 mL of the adsorption solution in the wells of the 24-well plate in Example 1) was sealed in a tube with a lid (5 mL assist tube), and an adsorption experiment was performed under sealed conditions.

Evaluation of base material (examination of hydrophilization treatment conditions)
The surface roughness (Sa) of the medical PE non-woven fabric used in this example and the surface area (A) per one side of the substrate (size 10 mm × 10 mm = 100 mm ² ) were examined in the same manner as in Example 1. It was 1.8 ± 0.2 μm, A = 201 ± 9 mm ² . It has been confirmed that the medical PE non-woven fabric also has a large surface roughness (about 3 times) and a surface area (about 2 times) as compared to the PS flat substrate.
The SEM image of each base-material surface before and behind plasma processing is shown in FIG. 5 left. At the strongest plasma processing conditions of 0.5 W / cm ² , a submicron-scale wavelike concavo-convex structure was formed, which was larger than the surface microstructure before plasma processing. For the other substrates, no significant structural change was observed as compared to before the plasma treatment. In addition, the contact angle of ultrapure water to the substrate surface (Fig. 5 right) was about 120 degrees before treatment, while all of the substrates after plasma treatment were reduced (about 40 degrees or less) From the above, the effect of improving the water wettability by the plasma treatment was confirmed. It is considered that the oxygen plasma treatment introduces a highly polar functional group such as an OH, CHO or COOH group to the hydrophobic PE surface. The water wettability improvement effect increased as the power density of plasma treatment increased from 0.1 to 0.5 W / cm ² .
After the adsorption treatment, the residual bFGF concentration in each adsorption solution and the bFGF concentration of an adsorption solution (without a substrate) held under the same conditions without immersion of the substrate were measured by ELISA. As shown in FIG. 6, the residual bFGF concentration in the adsorption solution after immersion of the untreated substrate (without plasma treatment) was approximately the same as the bFGF concentration in the adsorption solution retained without the substrate. It can be said that bFGF was hardly adsorbed to the untreated base material (PE non-woven fabric). On the other hand, in the plasma-treated substrate, the bFGF concentration in the adsorption solution was significantly reduced after immersion, and the adsorption of bFGF to the substrate was apparent. The amount of bFGF adsorbed to the plasma-treated substrate was calculated from 0.1 to 0.3 μg / cm ² when estimated from the decrease in bFGF concentration relative to the absence of the substrate. As a result of the improvement of the water wettability of the substrate by the plasma treatment, it is considered that the adsorption solution permeates the entire surface of the substrate and promotes the adsorption of bFGF on the substrate. Further, it is considered that the effect of the acidic functional group (such as COOH group) introduced to the substrate surface by plasma treatment promoting the interaction with bFGF which is a basic protein also contributes.

bFGF elution behavior (examination of hydrophilization treatment conditions)
The bFGF elution behavior from each substrate was examined in the same manner as Example 1. The bFGF elution behavior to the culture solution of each base material is shown in FIG. The untreated (no plasma treatment) substrate barely eluted bFGF, whereas all the plasma treated substrates released 3 ng / mL or more of bFGF slowly over 3 days. In addition, compared with the bFGF sustained-release porous substrate (bFGF adsorption type) prepared from PE non-woven fabric (thick) (thin) in Example 1 (FIG. 4 lower), it was prepared from PE non-woven fabric used in this example The amount of bFGF elution decreased with the substrate obtained. This is presumed to be due to the difference in the properties of the PE non-woven fabric and the change in the adsorption conditions (container, solution amount).
By hydrophilizing the substrate surface, it was confirmed that the amount of bFGF adsorbed to the substrate and the amount of bFGF eluted in the culture solution can be increased.

Preparation of bFGF sustained-release porous substrate (bFGF adsorption type) (adsorption treatment time, examination of the effect of shaking)
The PE non-woven fabric was washed in the same manner as in Example 2 (investigation of hydrophilization treatment), subjected to oxygen plasma treatment (0.1 W / cm ² ), and then sterilized with EOG gas. It was immersed in PBS (adsorbent) containing bFGF (4 μg / mL) at 25 ° C. for up to 48 hours. In the same manner as in Example 2 (investigation of hydrophilic treatment conditions), 1 mL of the adsorption solution was sealed in a tube with a lid (5 mL assist tube), and an adsorption experiment was performed under sealed conditions. At that time, when the tube with a lid is left standing in a cool incubator maintained at 25 ° C., and the tube with a lid is fixed in a shaking incubator (PIC-100S, manufactured by As One) kept at 25 ° C., and shaken (68 rpm, The case of 3 cm amplitude was examined.

Evaluation of base material (adsorption treatment time, examination of the effect of shaking)
After adsorption treatment for various times (0, 1, 3, 5, 8, 24, 48 hours), residual bFGF concentration in each adsorption solution and an adsorption solution (base material held under the same conditions without immersion) BFGF concentration of (none) was measured by ELISA. As shown in FIG. 8, the residual bFGF concentration in the adsorption solution decreased after treatment regardless of the presence or absence of the substrate, but the decrease width when the substrate was immersed (with the substrate) was smaller than that without the substrate. Was great. It is considered that the bFGF concentration decreases with the treatment time (̃8 hours) because a part of bFGF is degraded in the adsorption solution (PBS) when there is no substrate. On the other hand, when the substrate is immersed, it is considered that the residual bFGF concentration in the adsorption solution is further reduced by the adsorption of bFGF to the substrate in addition to the decomposition. When the amount of bFGF adsorbed to the substrate was estimated from the decrease in bFGF concentration relative to the residual bFGF concentration without the substrate, the amount of bFGF adsorbed to the substrate increased with the adsorption treatment time, and almost reached equilibrium in 24 hours . In addition, when the adsorption solution was shaken during the adsorption treatment (FIG. 8 lower), it was faster to reach equilibrium than when it was allowed to stand (upper FIG. 8). That is, it can be said that bFGF can be efficiently adsorbed onto the substrate if the adsorption solution is shaken. It is considered that this is because the adsorption solution reaches the inside of the porous substrate more quickly by shaking, and bFGF reduced near the surface by adsorption to the substrate is quickly compensated by the permeation and diffusion of the adsorption solution.

Preparation of bFGF sustained-release porous substrate (bFGF adsorption type) (examination of bFGF concentration of adsorption solution)
The base material was washed in the same manner as in Example 2 (investigation of hydrophilization treatment), subjected to oxygen plasma treatment (0.1 W / cm ² ), and then sterilized with EOG gas. The same substrate is immersed in PBS (adsorbent solution) to which bFGF is added at various concentrations (0, 0.1, 0.2, 0.5, 1, 2, 4, 8, 12 μg / mL) at 25 ° C. for 24 hours, and then washed. did. During immersion, a tube with a cap (5 mL assist tube) was fixed in a shaking incubator maintained at 25 ° C. and shaken as in the shaking conditions in Example 2 (adsorption treatment time, examination of the effect of shaking).

Evaluation of base material (examination of bFGF concentration of adsorption solution)
After the adsorption treatment, the residual bFGF concentration in each adsorption solution and the bFGF concentration of an adsorption solution (without a substrate) held under the same conditions without immersion of the substrate were measured by ELISA. The results are shown in FIG. When the amount of bFGF adsorbed to the substrate was estimated from the decrease in bFGF concentration relative to the residual bFGF concentration without the substrate, the amount of bFGF adsorbed to the substrate increased as the initial bFGF concentration in the adsorbent increased. However, when the initial bFGF concentrations were 8 μg / mL and 12 μg / mL, almost no difference was observed in the amount of bFGF adsorbed to the substrate, so it is considered that equilibrium is reached at about 8 μg / mL.

bFGF elution behavior (examination of bFGF concentration in adsorbed solution)
The bFGF elution behavior from each substrate was examined in the same manner as Example 2 (examination of hydrophilization treatment conditions). FIG. 10 shows the bFGF elution behavior to the culture solution of each substrate. As the initial bFGF concentration in the adsorption solution increased, the elution amount into the culture solution increased. Together with the results in FIG. 9, it was shown that when the initial bFGF concentration in the adsorption solution is high, the amount of bFGF adsorbed to the substrate increases, and the amount of bFGF eluted in the culture solution also increases. When the initial bFGF concentration of the adsorption solution is 8 μg / mL and 12 μg / mL, bFGF of 10 ng / mL or more can be sustained-released over 3 days in the culture solution, and 5 ng / mL or more can be sustained-released at 4 μg / mL. As described above, various bFGF elution behaviors can be obtained by appropriately setting the bFGF concentration of the adsorption solution, so that it is possible to prepare a bFGF sustained-release porous base material (bFGF adsorption type) according to the purpose. It can be said that there is.

Example 3 Examination of Substrate in Coprecipitation Method The coprecipitation method was used to form a bFGF-calcium phosphate composite layer on various porous substrate surfaces. The amount of bFGF eluted equal to or higher than that of Example 1 (adsorption method) was confirmed.

Preparation of bFGF sustained-release porous substrate (bFGF-calcium phosphate composite layer type) PE non-woven fabric (thin), PE non-woven fabric (thick), PP filter (pores), PP filter (large pore) used in Example 1 In addition to the PS flat substrate, a PP net filter (screen) (pore diameter 25 μm, thickness 360 μm, PP 2504700, manufactured by Merck Millipore) was newly prepared. The PP net filter was cut into 10 mm × 10 mm pieces and used as a substrate (without a washing step). Each substrate was treated with oxygen plasma in the same manner as in Example 1 and then sterilized with EOG gas. The plasma power density to the PP net filter was 0.5 W / cm ² .
A calcium phosphate supersaturated solution (hereinafter referred to as a supersaturated solution) to which bFGF (1 mg / mL, manufactured by Katayama Chemical Industries, Ltd.) is added so as to be 4 μg / mL, has been reported (H. Tsurushima et al. Acta Biomaterialia 6, 2751 (2010); , According to reference 1). 2 mL of the supersaturated solution was dispensed into a 24-well plate, and each substrate was immersed in the solution at 25 ° C. for 24 hours (coprecipitation method). At this time, the plasma-treated surface was placed in contact with the supersaturated solution (porous substrate: treated surface is down, PS flat substrate: treated surface is up). The 24-well plate was placed in a cool incubator kept at 25 ° C. in a zippered plastic bag (Unipack) to prevent evaporation of the supersaturated solution. After being immersed in the supersaturated solution, the substrate was removed from the solution and washed three times (1 second) with 2 mL of PBS (without bFGF added).

Evaluation of substrate The surface roughness (Sa) of the PP net filter added in this example and the surface area per one side of the substrate (A) were examined in the same manner as in Example 1. Sa = 75.2 ± 9.5 μm, A = It was 2398 ± 79 mm ² . The PP net filter was confirmed to have extremely large surface roughness (about 160 times) as well as surface area (about 24 times) as compared to PS flat substrate.
The SEM image of each base-material surface after a supersaturated solution process is shown in FIG. With any of the substrates, precipitates were observed on part of the surface after treatment. From the previous report (Reference 1), the precipitate is considered to be a bFGF-calcium phosphate composite layer formed by coprecipitation of calcium phosphate and bFGF.

bFGF elution behavior The bFGF elution behavior from the bFGF sustained-release porous substrate (bFGF-calcium phosphate composite layer-forming type) prepared by this coprecipitation method was examined in the same manner as Example 1. The left side of FIG. 12 shows bFGF elution behavior to cultures of three types of PP base and PS base. For PP filters (pores and large pores), bFGF sustained release equivalent to that of the bFGF sustained release porous substrate (bFGF adsorbed type) (FIG. 4 bottom) prepared by the adsorption method in Example 1 was confirmed. The The bFGF elution amount was lowest in the PS substrate, and then increased in the order of PP net filter <PP filter (large pore) <PP filter (pore). This is presumed to be due to the increase in the surface area (see Example 1) of the substrate due to the microscale uneven structure on the filter surface (FIG. 12 right). However, PP net filters, which had the largest effect of increasing surface roughness and surface area, had less bFGF elution than the other filters. This filter has a large fiber diameter (100 μm or more) and a thick base (0.5 mm or more) as shown in the SEM image at the upper right of FIG. Since the filter floats on water, it is considered that, although the effect of increasing the surface roughness and surface area of the whole substrate is large, the effective surface area which can actually contact the adsorption solution and the culture solution is small. In order to achieve the desired bFGF sustained release performance with a substrate suspended in water, it was considered desirable to have a surface micro-scaled surface relief structure.
FIG. 13 shows bFGF elution behavior to cultures of PP, PE, and PS substrates. Not only the PP substrate but also the PE substrate is equivalent to the bFGF sustained-release porous substrate (bFGF adsorption type) (FIG. 4 lower) prepared by the adsorption method in Example 1 depending on its fine structure, or It was confirmed that more bFGF was eluted.
From the above, similarly to the adsorption method of Example 1, also in the present coprecipitation method, when a PS base material having a dense and smooth surface is used by using a porous base material having large surface roughness and surface area. In comparison, it was confirmed that the bFGF elution amount can be increased. Moreover, it was confirmed that a base material having bFGF sustained release performance suitable for pluripotent maintenance culture of pluripotent stem cells can be produced by using a base material having an appropriate surface roughness and surface area.

Example 4 Investigation of Supersaturated Solution in Coprecipitation Method Various supersaturated solutions were used to form a bFGF-calcium phosphate composite layer on the substrate surface. In this example, a flat plate-like PS base material which is easy to handle although it sinks in water was used. The amount of bFGF eluted from the substrate could be controlled by the supersaturated solution and the conditions of treatment (temperature, composition, concentration of bFGF added).

Preparation of bFGF Sustained-Release Base Material (bFGF-Calcium Phosphate Composite Layer-Forming Type) In the same manner as in Example 1, a PS plate-like base material was prepared and subjected to oxygen plasma treatment. Subsequently, a bFGF-calcium phosphate composite layer was formed on the substrate surface using four known supersaturated solutions. The resulting substrate is named as follows.

・ Concentrated-low temperature-F4 (bFGF 4 μg / mL, 25 ° C.) Reference 1 (generally called RKM solution) same conditions as Example 3 Concentrated-low temperature-F10 (bFGF 10 μg / mL, 25 ° C.) Reference 1 (common name RKM) liquid)
・ Concentrated-high temperature-F10 (bFGF 10 μg / mL, 37 ° C.) Reference 2 (generally called RKM solution)
・ Thin-low temperature-F10 (bFGF 10 μg / mL, 25 ° C.) Reference 3 and 4 (generally called CP solution)

References 1 to 4 are as follows.
Reference 1 H. Tsurushima et al. Acta Biomaterialia 6, 2751 (2010).
Reference 2 H. Mutsuzaki et al. J Biomed Mater Res B, 86B, 365 (2008).
Reference 3 M. Uchida et al. Adv Mater 16, 1071 (2004).
Reference 4 K. Sasaki et al. Biomed Mater 5, 065008 (2010).

The supersaturated solutions described in references 1 and 2 commonly referred to as RKM solutions contain high concentrations of calcium phosphate component ions and carbonate ions. It is also an unstable supersaturated solution that induces uniform nucleation of calcium phosphate within 24 hours of preparation because the solution pH increases with time (due to decarboxylation). On the other hand, the supersaturated solution described in Reference 3 generally called CP solution has a lower concentration of calcium phosphate component ions than that of the RKM solution, and the pH buffer has less fluctuation in pH. For this reason, it is a metastable supersaturated solution that does not induce homogeneous nucleation over several weeks after preparation.
The concentrated-low temperature-F4, concentrated-low temperature-F10, and concentrated-high temperature-F10 were prepared by sterilizing the substrate after plasma treatment with EOG gas as in Example 3 and following the previous report (references 1 and 2) C. or 37.degree. C. for 24 hours in 1 mL of the supersaturated solution prepared above. In thin-low temperature F10, after the substrate after plasma treatment is subjected to alternate immersion treatment (Reference 4) and precoated with calcium phosphate, 1 mL of the supersaturated solution prepared according to the previous report (references 3 and 4) It was prepared by immersion at 24 ° C. for 24 hours. After being immersed in the supersaturated solution, the substrate was removed from the solution and washed three times (1 second) with 2 mL of PBS (without bFGF added).

Substrate Evaluation FIG. 14 shows SEM images of the substrates after various supersaturated solution treatments. A deposit was confirmed on the surface of the substrate after treatment. From the previous report (References 1 to 4), the precipitate is considered to be a bFGF-calcium phosphate composite layer formed by coprecipitation of calcium phosphate and bFGF.

bFGF elution behavior The bFGF elution behavior from the bFGF sustained-release PS base material (bFGF-calcium phosphate composite layer-formed type) prepared using various supersaturated solutions was examined in the same manner as Example 1. However, 2 mL of culture solution was placed in a screw-cap tube (5 mL assist tube) instead of the 24-well plate, and the elution experiment was performed under closed conditions. A calibration curve for measuring the bFGF concentration of the eluate was prepared using the standard bFGF attached to the ELISA kit.
As shown in FIG. 15, the bFGF elution amount was different depending on the conditions of the supersaturated solution. When the same supersaturated solution (the RKM solution in reference 1) is used, the substrate with higher concentration of bFGF added to the supersaturated solution (thick-low temperature-F10) has lower substrate (high-low temperature-F4) More bFGF was eluted. Among the four supersaturated solutions used in this example, the amount of bFGF eluted from the substrate (thin-low temperature-F10) produced using the CP solution of References 3 and 4 was the smallest. This is because the loading efficiency of bFGF, which is a basic protein, is low in CP solution (A. Oyane et al. Acta Biomaterialia 7, 2969 (2011); hereinafter referred to as Reference 5), only a small amount of bFGF is the substrate surface. Probably because it was not supported by
In the coprecipitation method, various supersaturated solution conditions (temperature, composition, bFGF added concentration) can be used, and it was confirmed that the amount of bFGF eluted from the substrate can be controlled by selecting these conditions.

Example 5 Examination of Substrate and Hydrophilization Treatment in Coprecipitation Method The bFGF-calcium phosphate composite layer was formed on the surface of the mesh substrate and the porous substrate using the coprecipitation method. It was confirmed that a large surface area porous substrate can elute more bFGF than a large gap mesh structure, and that the amount of bFGF eluted can be increased by hydrophilization pretreatment (plasma treatment) to the substrate .

Preparation of bFGF sustained-release base material (bFGF-calcium phosphate composite layer-forming type) The following PE mesh and porous sheet were cut into 10 mm × 10 mm pieces and used as a base material (no washing step) (FIG. 16) ). A PS flat substrate for comparison was prepared in the same manner as in Example 1.

・ PE mesh: PE 200 (made by NBC Meshtec) opening 112 μm
・ PE porous sheet: Sun map LC-T (made by Nitto Denko)

Each substrate was subjected to oxygen plasma treatment (0.5 W / cm ² , 30 Pa, 13.56 MHz, 30 seconds) in the same manner as in Example 1. The untreated (without plasma treatment) and plasma-treated substrates were sterilized with EOG gas and immersed in 2 mL of a supersaturated solution containing bFGF (4 μg / mL) at 25 ° C. for 24 hours as in Example 3 and washed. . At this time, the surface on which the bFGF-calcium phosphate composite layer was formed (plasma-treated surface) was placed in contact with the supersaturated solution (PE base: treated side is lower, PS base: treated side is upper).

Evaluation of Substrates The surface roughness (Sa) of each substrate and the surface area (A) per surface of the substrate (size 10 mm × 10 mm = 100 mm ² ) were examined in the same manner as in Example 1. The results are shown below (numbers are the mean of three measurements ± standard deviation).

PS flat substrate Sa = 0.5 ± 0.0 μm, A = 109.3 ± 1.1 mm ²
PE mesh sheet Sa = 87.3 ± 10.1 μm, A = 2339 ± 312 mm ²
PE porous sheet Sa = 11.7 ± 0.8 μm, A = 595 ± 36 mm ²

When ultrapure water was dropped on the substrate surface before and after the plasma treatment, it was confirmed that the water wettability of the substrate surface is improved by the plasma treatment for any substrate.
FIG. 17 shows an SEM image of the surface of the substrate (without plasma treatment) after the supersaturated solution treatment. A deposit was confirmed on the surface of the substrate after treatment. From the previous report (Reference 1), the precipitate is considered to be a bFGF-calcium phosphate composite layer formed by coprecipitation of calcium phosphate and bFGF.

bFGF elution behavior The bFGF elution behavior from the prepared bFGF sustained-release base material (bFGF-calcium phosphate composite layer formed type) was examined in the same manner as in Example 1 (PE base material: treated side down, PS base material: Processing side is top). However, a standard curve for measuring the bFGF concentration of the eluate was prepared using the standard bFGF attached to the ELISA kit.
As shown in FIG. 18, the amount of bFGF elution was larger in the substrate (lower) subjected to plasma treatment than in the substrate (upper) not subjected to plasma treatment. It is considered that this is because the amount of bFGF loaded on the substrate surface is increased by plasma treatment. It was found that in the coprecipitation method as well as the adsorption method (Example 2), the hydrophilization of the substrate surface by plasma treatment was effective in improving the bFGF elution amount. Further, when compared with the plasma-treated substrate (FIG. 18 lower), in the mesh substrate, the bFGF elution amount can hardly be increased relative to the PS flat substrate having a smooth surface, while the porous substrate is porous. In the substrate, the bFGF elution amount could be increased. The mesh base used in this example has a large fiber diameter (about 100 μm), a thick base, and a large opening (a gap between fibers) (FIG. 16 upper left). Therefore, although the effect of increasing the surface roughness and surface area of the whole substrate is increased, the PE substrate floats on water, so it is considered that the effective surface area which can actually contact the adsorption liquid and the culture solution is small. The above also corresponds to the tendency observed in Example 3.

Example 6 Examination of bFGF Concentration and Heparin Addition in Adsorption Method Using an adsorption solution to which bFGF and heparin were added at various concentrations, bFGF was adsorbed to a hydrophilic membrane filter. It was confirmed that the elution amount of bFGF into the culture solution increased according to the bFGF and heparin addition concentration.

Preparation of bFGF sustained release substrate (bFGF adsorption type) As described below, membrane filters for filtration made of hydrophilized polyvinylidene fluoride (PVDF) and cellulose mixed ester are cut into 10 mm × 10 mm sections, and (Figure 19). Although any base material sinks in water, it was used in this example for examination of bFGF concentration and heparin addition effect.

-PVDF: Durapore DVPP 04700 (manufactured by Merck Millipore), pore diameter 0.65 μm, thickness 125 μm
Ester: MF-Millipore GSWP 04700 (Merck Millipore), pore diameter 0.22 μm, thickness 150 μm

The final concentration of bFGF is 0.1, 1.0 μg / mL in PBS, heparin sodium preparation (100 units / mL syringe for heparin Na lock “Otsuka” 5 mL, Otsuka Pharmaceutical), final concentration 0, 0.1, 1, An adsorption liquid was prepared by adding 10 units / mL. In the same manner as in Example 2, each base material was sterilized with EOG gas, immersed in 1 mL of each adsorption solution at 25 ° C. for 6 hours, and washed.

Evaluation of Substrates The surface roughness (Sa) of each substrate and the surface area (A) per surface of the substrate (size 10 mm × 10 mm = 100 mm ² ) were examined in the same manner as in Example 1. The results are shown below (numbers are the mean of three measurements ± standard deviation).

PVDF Sa = 0.68 ± 0.01 μm, A = 229.6 ± 1.8 mm ²
Ester Sa = 0.16 ± 0.01 μm, A = 150.9 ± 1.8 mm ²

bFGF elution behavior The bFGF elution behavior from the prepared bFGF sustained release substrate (bFGF adsorption type) was examined in the same manner as Example 1. However, the amount of culture solution dispensed into the wells of the 24-well plate was 1 mL. In addition, the collection amount of the eluate was 100 μL, and a calibration curve for measuring the bFGF concentration of the eluate was prepared using the standard bFGF attached to the ELISA kit.
The bFGF elution behavior of the PVDF substrate is shown in FIG. 20, and the bFGF elution behavior of the ester substrate is shown in FIG. The elution amount of bFGF increased with the increase of the heparin concentration in the adsorption solution for any base material and any bFGF concentration. It is considered that this is because the heparin binding protein bFGF binds to and stabilizes heparin in the adsorption solution (and the culture solution).
In addition, for any of the substrates, when the bFGF concentration in the adsorption solution is high (FIG. 20, bottom: 1.0 μg / mL), it is lower than when it is low (FIG. 20, top: 0.1 μg / mL) The amount of bFGF elution increased about 10 times. This is considered to be because the bFGF adsorption amount to the base material increases in accordance with the bFGF concentration in the adsorption solution.
It was confirmed that the amount of bFGF eluted in the substrate can be controlled by adjusting the concentration of bFGF and heparin added in the adsorption solution.

Example 7 Examination of Heparin Addition in Coprecipitation Method A bFGF-calcium phosphate composite layer was formed on the surface of a PS substrate using a supersaturated solution to which heparin was added at various concentrations. It was confirmed that the amount of bFGF eluted into the culture solution increased according to the heparin addition concentration.

Preparation of bFGF sustained-release base material (bFGF-calcium phosphate composite layer-forming type) Heparin sodium preparation (100 units / mL syringe for heparin Na lock "Otsuka" in bFGF (4 μg / mL) containing supersaturated solution used in Example 3 Three supersaturated solutions were prepared by adding 5 mL (manufactured by Otsuka Pharmaceutical Co., Ltd.) to a final concentration of 0, 0.1, 3.6 unit / mL. In the same manner as in Example 3, the PS plate-like substrate was sterilized with EOG gas, immersed in 2 mL of each supersaturated solution at 25 ° C. for 24 hours, and washed.

bFGF Elution Behavior The bFGF elution behavior from the prepared bFGF sustained-release base material (bFGF-calcium phosphate composite layer formed type) was examined in the same manner as Example 5. However, Dulbecco's modified Eagle's medium (DMEM, manufactured by Sigma Aldrich) was used as an eluate. As shown in FIG. 22, the amount of bFGF eluted from the substrate increased as the heparin concentration in the supersaturated solution increased. Similar to the results of Example 6 (adsorption method), also in the coprecipitation method, it was confirmed that addition of heparin to the treatment solution was effective in improving the bFGF elution amount.

Example 8 Examination of Substrate Form (Bead-Like Substrate) in Coprecipitation Method A calcium phosphate layer was formed on the surface of a bead-like PS substrate.

As a production base of a base material (calcium phosphate layer formation type), bead-like PS particles (SBX-17, manufactured by Sekisui Plastics Co., Ltd.) having a particle size of 17.08 ± 6.55 μm were prepared. Chemical treatment was carried out under the following two conditions in order to hydrophilize the substrate surface. Under the condition 1, 0.101 g of PS particles was immersed, and under the condition 2, 0.0195 g of PS particles was immersed in 10 mL of each treatment solution described below. The treatment liquid was sealed in a PP tube with a lid, and treated at 40 ° C. for a specified time under shaking (shaking bath BW101 made by Yamato) at a rotational speed of 150 rpm.

Condition 1: 1.0 M HCl aqueous solution, 10 minutes Condition 2: 1.0 M HCl 50 vol% aqueous ethanol solution, 60 minutes

Subsequently, alternate immersion treatment (Reference 4) was applied to pre-coat calcium phosphate on the substrate surface. However, in this example, in order to treat a large amount of bead-like PS base material floating in water, the treatment liquid (100 mM-CaCl ₂ 50% ethanol aqueous solution → 50% ethanol aqueous solution (washing liquid) → 100 mM-) using a suction filtration system. K ₂ HPO ₄ 50% ethanol aqueous solution → 50% ethanol aqueous solution (washing solution), the above cycle is repeated 3 times) in contact with the substrate in the specified order.
After alternate immersion treatment, the substrate is air-dried overnight, and suspended in approximately 15 mL of condition 1 and 7 mL of a supersaturated solution (generally called CP solution (References 3 and 4) under condition 2) to obtain bead-like PS. A suspension of the substrate was obtained. This suspension was diluted 1, 3, or 10 times to a 5 mL supersaturated solution. The same supersaturated solution was sealed in a capped PP tube, and treated at a temperature of 25 ° C. under shaking under the following conditions. In condition 1, processing was performed at rotational speed 3 (small shaking incubator PIC-100S, manufactured by As One) for 48 hours, and in condition 2, processing was performed at rotational speed 200 rpm (Bioshaker BR-42FM, manufactured by Taitec) for 23 hours.

Evaluation of Substrate A SEM image of the surface of the substrate after supersaturated solution treatment is shown in FIG. A deposit was confirmed on the surface of the substrate after treatment. From the previous report (Reference 1), the precipitate is considered to be a calcium phosphate layer. The coverage of the calcium phosphate layer improved with the increase in dilution ratio to the supersaturated solution, and the entire surface of the substrate was coated with the calcium phosphate layer under the 10-fold dilution condition. By increasing the dilution ratio, it was thought that the amount of liquid phase relative to the solid phase was increased, facilitating the contact of the substrate with the supersaturated solution and the supply of raw materials (such as calcium ions, phosphate ions and their clusters). .
By using this method, it was confirmed that a calcium phosphate layer can be formed not only on the flat sheet, film, and substrate used in the previous example but also on the surface of a bead-like substrate. As shown in Examples 3 to 6, the bFGF-calcium phosphate composite layer can be formed on the surface of the bead-like substrate by adjusting the kind of supersaturated solution, bFGF added concentration, heparin added concentration, etc. It is considered that the same base material can be provided with bFGF sustained release ability.

(Example 9) Functional demonstration of bFGF sustained-release porous substrate (bFGF adsorption type)
It was confirmed that human iPS cells can be cultured for undifferentiated maintenance in a culture solution in which bFGF sustained-release porous base material (bFGF adsorption type) is placed (floating in the culture solution).

Preparation of bFGF sustained-release porous base material (bFGF adsorption type) The base material was washed and treated with oxygen plasma (0.1 W / cm ² ) in the same manner as in Example 2 (examination of hydrophilization treatment conditions). Then, it was sterilized with EOG gas. The same base material is immersed in PBS (adsorption solution) to which bFGF (12 μg / mL) is added in the same manner as in Example 2 (examination of bFGF concentration of the adsorption solution) at 25 ° C. for 24 hours. Washed with). During immersion, a tube with a cap (5 mL assist tube) was fixed in a shaking incubator maintained at 25 ° C. and shaken as in the shaking conditions in Example 2 (adsorption treatment time, examination of the effect of shaking).

iPS cell culture human iPS cells (strain 201B7) were distributed from RIKEN BioResource Center. Positive culture conditions in a normal culture solution (Essential 6 Medium (Thermo Fisher Scientific), 2 ng / mL TGF-β 1 (R & D Systems), 10 ng / mL bFGF (Katayama Chemical)) to which bFGF was added The control condition (2), the culture condition obtained by removing the bFGF from the culture solution of the condition (2) was taken as a negative control (condition (3)), and the bFGF sustained-release porous substrate (bFGF adsorption type) was used instead of bFGF. The comparison with the culture conditions (condition (1)) in the culture solution in which was placed was carried out. For culture, 6 wells of culture dishes were used, and each well was coated with Matrigel basement membrane matrix growth factor reduce (manufactured by Corning), and the volume of the culture solution was 2 mL.
First, human iPS cells were seeded on a culture dish (this time is hereinafter referred to as culture day 0), and preculture was performed for 1 day under normal culture conditions (same as condition (2)). Immediately after passage on the 4th day of culture, preculture was performed for 1 day under normal culture conditions (same as the condition (2)). In condition (1), bFGF sustained-release porous substrate (bFGF adsorption type) was placed on the 1st and 5th day of the culture, and 3 days (up to the 4th day and 8 days) without culture fluid exchange. Cultured to the eye). In the conditions (2) and (3), the culture solution ((2): containing bFGF, (3): no bFGF) was exchanged daily on the first to third days of culture and on the fifth to seventh days of culture. .

Measurement of bFGF concentration in culture solution under human iPS cell culture The bFGF concentration in culture solution on the 4th and 8th day of culture under each condition ((1), (2), (3)) was measured by ELISA . For comparison, a bFGF-free culture solution (condition (4): a system obtained by removing cells from condition (1)) in which bFGF sustained-release porous substrate (bFGF-adsorbed type) is placed without cells is also included. , ELISA measurements were performed. A calibration curve for measuring bFGF concentration was prepared using bFGF (manufactured by Katayama Chemical Industry Co., Ltd.).
FIG. 24 shows bFGF concentrations in the culture solution on the 4th and 8th day of culture under each condition. The bFGF concentration in the culture solution under cell-free conditions (4) is about 15 to 17 ng / mL, and the same experimental system (FIG. 10 of Example 2, the size and surface treatment conditions of the wells The same bFGF concentration was confirmed as the bFGF concentration (about 12 ng / mL after 3 days) measured in different). On the other hand, bFGF is hardly detected in the culture solution under the condition (3) (bFGF without culture solution, culture solution exchanged every day) which is a negative control, and normal culture conditions (2) The bFGF concentration in the culture solution of 2) was about 2 ng / mL. Under normal culture conditions (2), the culture solution containing 10 ng / mL bFGF is exchanged daily, but the bFGF concentration in the culture solution detected by ELISA one day after the exchange is only about one fifth of the initial addition concentration It was confirmed that there was not. On the other hand, under the condition (1) (culture solution provided with bFGF sustained release porous base material (bFGF adsorption type), without culture solution exchange), a high bFGF concentration of 20 ng / mL or more is maintained even after 3 days It had been. Under the condition (1), the bFGF concentration was higher than the culture solution of the condition (4) containing no cells, and it was presumed that bFGF secreted from the cells was added.

Evaluation of undifferentiated nature of human iPS cells 1 (morphological observation)
The morphology of human iPS cells cultured in each condition was observed by phase contrast microscopy. FIG. 25 shows phase contrast microscopic images of human iPS cells on days 1, 4, 5, and 8 of culture. Human iPS cells cultured in the negative control condition (3) (culture medium without bFGF, culture medium changed every day) open the intercellular space on days 4 and 8 of culture (day 4 after passage) The colonies formed only irregular shapes, and about half of the cells deviated from the colonies (FIG. 25 (3)). On the other hand, human iPS cells cultured under the normal culture condition (2) (the culture solution containing bFGF, and the culture solution exchanged every day) which is a positive control are the fourth and eighth days of culture (the fourth day after passage) Colonies of normal form (FIG. 25 (2)). The iPS cells cultured under the condition (1) (a culture solution provided with bFGF sustained-release porous substrate (bFGF adsorption type), without culture solution exchange) also showed similar colony formation of normal morphology ((1) FIG. 25 (1). From this, it was demonstrated that even under the condition (1), human iPS cells can be cultured as in the normal culture condition (2).

Evaluation of undifferentiated property of human iPS cells 2 (cell staining)
The cells are fixed with 4% paraformaldehyde on the 7th day of culture, washed with PBS, and then undifferentiated by rBC2LCN lectin staining and fluorescence labeled antibody (Nanog, Oct3 / 4) staining, which is a general diagnostic method for human iPS cells. The sex was evaluated. In rBC2LCN staining, rBC2LCN (manufactured by Wako Pure Chemical Industries, Ltd.) directly labeled with FITC was reacted with the cells, and a stained image was obtained by a fluorescence microscope. In fluorescent antibody staining, after reacting with an anti-Nanog antibody (manufactured by Cell Signaling) or an anti-Oct3 / 4 antibody (manufactured by Santa Cruz), a secondary antibody anti-mouse IgM-Alexa 488 (manufactured by Thermo Fisher Scientific) or an anti -Mouse IgG-Alexa 488 (manufactured by Thermo Fisher Scientific) was further reacted, and a stained image was obtained by a fluorescence microscope.
FIG. 26 shows fluorescence microscopic images of human iPS cells stained by each method. Staining of human iPS cells cultured under normal culture conditions (2) (culture medium with bFGF, culture medium replaced daily) was confirmed by any staining method of rBC2LCN staining and antibody staining (FIG. 26 (2)) . Similar staining was also confirmed in human iPS cells cultured under the condition (1) (a culture solution provided with bFGF sustained-release porous base material (bFGF adsorption type), without culture solution exchange) (FIG. 26 (1 )). From this, it was demonstrated that even under the condition (1), the expression of the undifferentiated marker of human iPS cells can be maintained as in the normal culture condition (2).

  From the above results, instead of adding bFGF to the culture solution, bFGF sustained-release porous base material (bFGF adsorption type) is placed in the culture solution to add bFGF to the culture solution or change the culture solution daily It has been confirmed that undifferentiated maintenance culture of human iPS cells can be performed even if not performed.

(Example 10) Examination of liquid factor in adsorption method The adsorption method is also effective for liquid factors other than bFGF, and it is possible to produce various liquid factor sustained-release porous substrates (liquid factor adsorption type) It was confirmed.

Selected as a humoral factor that dissolves in a medium for selection of humoral factors and affects cell growth, differentiation, etc., has a molecular weight similar to that of bFGF (Recombinant Human bFGF, molecular weight = 16 kDa, manufactured by Katayama Chemical Industry Co., Ltd.). The following two types of proteins were selected.

・ Macrophage colony stimulating factor (M-CSF)
・ Tumor necrosis factor α (TNF α)

Both the Recombinant Human M-CSF and Recombinant Human TNF [alpha] made ORF Genetics used in this example (ISOkine ^TM) is equipped with Histidine-based tag, a molecular weight, respectively 20.7kDa and 19.6KDa.

Preparation of liquid factor sustained-release porous base material (liquid factor adsorption type) The base material (PE non-woven fabric) was washed and oxygen plasma treated in the same manner as in Example 2 (investigation of the hydrophilization treatment conditions) After applying 0.1 W / cm ² ), it was sterilized with EOG gas. The same substrate is immersed in PBS (adsorbent solution) to which M-CSF or TNFα (4 μg / mL) has been added in the same manner as in Example 2 (examination of bFGF concentration in the adsorbate) for 24 hours at 25 ° C. Washed with (no bFGF added). During immersion, a tube with a cap (5 mL assist tube) was fixed in a shaking incubator maintained at 25 ° C. and shaken as in the shaking conditions in Example 2 (adsorption treatment time, examination of the effect of shaking).

Solution factor elution behavior The elution behavior of M-CSF and TNFα from each substrate was examined in the same manner as in Example 1. However, the concentrations of M-CSF and TNFα in the eluate were measured using R & D ELISA kit (Quatikine ELISA Human M-CSF Immunoassay and Quatikine ELISA Human TNFα Immunoassay). In addition, standard curves were prepared using the standard M-CSF and TNFα attached to each ELISA kit.
The elution behavior of M-CSF and TNFα into the culture solution of each substrate is shown in FIGS. 27 and 28, respectively. As with the bFGF sustained-release porous substrate (bFGF-adsorbed type) (Fig. 10), the substrate to which any liquid factor is adsorbed shows the maximum concentration after one day, and then the concentration decreases with time. However, even after 7 days, the concentration was maintained at about half of the maximum concentration. The concentration of sustained-release humoral factor in the culture solution was different depending on the type of factor, and M-CSF was 7 to 8 times higher concentration than TNFα over the whole evaluation period up to 7 days later. In addition, when compared also including bFGF sustained release porous base material (bFGF adsorption type) (FIG. 10) prepared using the same concentration (4 μg / mL) of adsorption solution, the fluid which was released in the culture solution was compared The concentration of sexual factor increased in the order of TNFα <bFGF <M-CSF. The difference in sustained release concentration is considered to be due to the difference in the molecular structure (steric structure, composition, hydrophilicity / hydrophobicity, isoelectric point, etc.) of the liquid factor.

  As mentioned above, by adding other humoral factors (TNFα, M-CSF) to the adsorption solution instead of bFGF, the substrate which floats in the culture solution and which releases the same humoral factor into the culture solution is used. It confirmed that it could produce. Such a substrate is considered to be effective not only as an undifferentiated maintenance culture of undifferentiated cells but also as a culture solution additive for controlling cell proliferation and differentiation.

  According to the present invention, various humoral factors that affect the undifferentiated maintenance or differentiation / proliferation of mammalian cells can be slowly released into the cell culture medium, and as a result, these factors can be released over a long period of time. As the concentration in the culture medium can be maintained in the necessary range, in the field of medicine such as regenerative medicine and drug discovery, mammalian undifferentiated cells such as embryonic stem cells and iPS cells are maintained undifferentiated, It can be used when differentiating / proliferating to a target differentiated cell or tissue at a required time.

Claims (24)

  In a substrate having a porous structure at least the surface of which is hydrophilic, a humoral factor causing undifferentiated growth of mammalian undifferentiated cells, or a humoral factor affecting differentiation and proliferation of mammalian cells Adsorbed, cell culture supplement.   The substrate having a porous structure is a fiber assembly, a porous membrane, a substrate having a porous surface only, or a substrate having the porous substrate adhered to the surface. Cell culture supplements.   The cell culture support according to claim 1 or 2, wherein the substrate having a porous structure contains polyethylene, polypropylene or polystyrene as a material.   The cell culture supplement according to any one of claims 1 to 3, which has a specific gravity smaller than that of the cell culture solution.   The cell culture support according to any one of claims 1 to 4, wherein the liquid factor is a factor capable of binding to heparin, and a sulfated polysaccharide is further adsorbed on a substrate having a porous structure.   The cell culture supplement according to any one of claims 1 to 5, wherein the humoral factor is fibroblast growth factor-2 (bFGF).   It contains a humoral factor that hydrophilizes the surface of a substrate having a porous structure and causes it to proliferate undifferentiated cells of a mammal without differentiation, or contains a humoral factor that affects differentiation and proliferation of mammalian cells. The method for producing a cell culture supplement according to any one of claims 1 to 4 and 6, wherein the agent is adsorbed to a substrate having a porous structure by immersing in a solution.   A humoral factor that hydrophilizes the surface of a substrate having a porous structure and causes it to grow undifferentiated cells of a mammal without differentiation, or a humoral factor that affects differentiation and proliferation of mammalian cells, The cell culture supplement according to claim 5 or 6, wherein the factor and the sulfated polysaccharide are adsorbed to a substrate having a porous structure by immersing in a solution containing the sulfated polysaccharide. Production method.   The method for producing a cell culture support according to claim 7 or 8, wherein hydrophilization of the surface of the substrate having a porous structure is performed by plasma treatment or acid treatment.   A method of culturing a mammalian cell while introducing the cell culture supplement according to any one of claims 1 to 6 into a cell culture medium for culturing a mammalian cell, and releasing the liquid factor in the medium. .   The mammalian cell is an undifferentiated cell, the humoral factor is a humoral factor that causes the undifferentiated cell to grow in an undifferentiated state, and the undifferentiated cell is maintained in an undifferentiated state and cultured. The method according to claim 10.   11. The method according to claim 10, wherein the humoral factor is a humoral factor that causes mammalian cells to differentiate and grow into differentiated cells or tissues, and the mammalian cells are differentiated and grown into differentiated cells or tissues by culturing. Method.   A calcium phosphate layer containing a humoral factor that causes undifferentiated cells of a mammal to grow undifferentiated on at least the surface of a substrate having a hydrophilic surface, or a humoral factor that affects differentiation and proliferation of mammalian cells Cell culture supplement with.   The substrate is a substrate having a porous structure, which is a fiber assembly, a porous membrane, a substrate having a porous surface only, or a substrate having the porous substrate adhered to the surface. The cell culture supplement according to claim 13.   The cell culture supplement according to claim 13 or 14, wherein the substrate comprises polyethylene, polypropylene or polystyrene as a material.   The cell culture supplement according to any one of claims 13 to 15, which has a specific gravity smaller than that of the cell culture solution.   The cell culture supplement according to any one of claims 13 to 16, wherein the humoral factor is a heparin-binding factor, and the calcium phosphate layer further contains a sulfated polysaccharide.   The cell culture support according to any one of claims 13 to 17, wherein the humoral factor is fibroblast growth factor-2 (bFGF).   In a calcium phosphate supersaturated solution containing a humoral factor that hydrophilizes the surface of a substrate and causes undifferentiated growth of mammalian undifferentiated cells or a humoral factor that affects mammalian cell differentiation and proliferation The method for producing a cell culture supplement according to any one of claims 13 to 16 and 18, wherein the calcium phosphate layer containing the factor is formed on the surface of the substrate by immersion.   A humoral factor that hydrophilizes the surface of a substrate and causes undifferentiated growth of mammalian undifferentiated cells, or a humoral factor that affects mammalian cell differentiation and proliferation, and sulfated polysaccharide The method for producing a cell culture supplement according to claim 17 or 18, wherein the calcium phosphate layer containing the factor and the sulfated polysaccharide is formed on the substrate surface by immersing in a calcium phosphate supersaturated solution containing the method.   The method for producing a cell culture supplement according to any one of claims 19 or 20, wherein the surface of the substrate is hydrophilized by plasma treatment or acid treatment.   A method for culturing a mammalian cell while supplying the cell culture supplement according to any one of claims 13 to 18 to a cell culture medium for culturing a mammalian cell, and releasing the liquid factor into the medium in a controlled manner. .   The mammalian cell is an undifferentiated cell, the humoral factor is a humoral factor that causes the undifferentiated cell to grow in an undifferentiated state, and the undifferentiated cell is maintained in an undifferentiated state and cultured. The method according to claim 22,.   The humoral factor is a humoral factor that causes mammalian cells to differentiate and proliferate mammalian cells into differentiated cells or tissues, and is characterized in that mammalian cells are differentiated and proliferated into differentiated cells or tissues by culturing. Method.

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