JP2010158180A - Scaffold material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a scaffold material using a synthetic polymer for culturing undifferentiated cells having pluripotency, such as ES cells, AS cells, iPS cells and the like, in the undifferentiated state. <P>SOLUTION: The scaffold material for culturing undifferentiated cells in the undifferentiated state comprises a hydrogel on which undifferentiated cells, such as ES cells, adult stem cells, iPS cells and the like, are cultured in the undifferentiated state. On the hydrogel, the undifferentiated cells are cultured in the undifferentiated state, wherein oxygen and nutrients are supplied sufficiently and efficiently to the undifferentiated cells without a problem of viral infection, contacting with the cells of different species or the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a scaffold material for culturing undifferentiated cells having multipotency such as embryonic stem cells, adult stem cells, and human induced pluripotent stem cells in an undifferentiated state.

  In recent years, techniques and organisms for separating undifferentiated cells such as embryonic stem cells (ES cells), adult stem cells (Adult Stem cells; AS cells), and induced pluripotent stem cells (iPS cells). Undifferentiated cells through the development of technology for inducing differentiation into tissue cells, bio-printing technology for rapid prototyping of biological tissues and tissue regeneration scaffolds, or biomaterial scaffold materials for regenerating more complex cells It is possible to produce biological tissue from Since these undifferentiated cells are cells having pluripotency such as pluripotency and pluripotency, particularly the application of human undifferentiated cells in the field of regenerative medicine and tissue engineering is expected. Clinical trials and early commercialization are being conducted.

  Therefore, in recent years, researches for controlling differentiation from undifferentiated cells to tissue cells have been actively conducted. For example, mouse ES cells maintain undifferentiation by co-culturing on feeder cells such as mouse embryonic fibroblast (MEF) in the presence of leukemia inhibitory factor (LIF). It has been confirmed that this is possible (Non-Patent Document 1). Further, it has been confirmed that human ES cells can maintain undifferentiation in the presence of bFGF (Basic Fibroblast Growth Factor) and heparin (Non-patent Document 2).

  However, human ES cells, human AS cells, human iPS cells and the like are cells that are easily differentiated and difficult to be passaged while maintaining a pluripotent state. In order to use it in the field of tissue engineering, a technique for culturing a large amount in an undifferentiated state is necessary, but an optimal culture method capable of performing this has not been established yet.

  On the other hand, the present inventors have created various regenerative medical materials and tissue engineering materials by applying a hydrogel that was originally developed. For example, Japanese Patent Application Laid-Open No. 2006-42795 discloses a cell culture containing a synthetic polymer having a monomer unit of sodium p-styrenesulfonate (NaSS) or sodium 2-acrylamido-2-methylpropanesulfonate (NaAMPS). A base material is disclosed (Patent Document 1), and Japanese Patent Application Laid-Open No. 2007-307300 discloses poly (sodium p-styrenesulfonate) (PNaSS) gel or poly (2-acrylamido-2-methyl-1-propane). An artificial blood vessel material which is a sodium sulfonate (PNaAMPS) gel is disclosed (Patent Document 2).

JP 2006-42795 A JP 2007-307300 A

Smith A.G. et al., Nature 336: 688-90 (1988) Miho K. et al., Proc. Natl. Acad. Sci. USA 105 (36), 13409-13414 (2008)

  Based on the cell culture substrate and artificial blood vessel material disclosed in Patent Document 1 and Patent Document 2, it is impossible to establish a scaffold material that can culture undifferentiated cells in an undifferentiated state. Culture methods are needed.

  The present invention has been made in order to solve the above-mentioned problems, and is a synthetic compound for culturing undifferentiated cells having multipotency such as ES cells, AS cells, iPS cells and the like in an undifferentiated state. The object is to provide a scaffold material using molecules.

  The scaffold material according to the present invention is a scaffold material for culturing undifferentiated cells in an undifferentiated state, and is made of hydrogel.

  In a preferred embodiment of the present invention, the hydrogel has 2-acrylamido-2-methylpropanesulfonic acid (AMPS), p-styrenesulfonic acid (SS), or a metal or base added to the sulfonic acid group. Hydrogel containing salt as monomer unit, hydrogel containing N, N-dimethylacrylamide (DMAAm) as monomer unit, hydrogel containing vinyl alcohol (VA) as monomer unit, or hydrogel containing two or more kinds of monomer units The scaffold material is a hydrogel selected from the group consisting of:

  According to the present invention, oxygen and nutrients can be sufficiently and efficiently supplied to undifferentiated cells without causing problems such as virus infection and contact with heterologous cells, and undifferentiated cells can be removed on a hydrogel. It can be cultured in a differentiated state.

It is a photographic diagram showing the morphology of mouse ES cell colonies in a cross-link density of 0.5 mol% PNaAMPS discs (24 hours, 48 hours, 72 hours in order from left to right, 96 hours, 120 hours in order from left to right). . It is a photograph showing the morphology of mouse ES cell colonies (48 hours, 72 hours, 96 hours in order from the upper left to the right, 120 hours in the order from the lower left to the right, 168 hours in the order from the lower left to the right) on the 2 mol% PNaAMPS disk with a crosslinking density. It is a photographic diagram showing the morphology of mouse ES cell colonies (48 hours, 72 hours, 96 hours in order from the upper left to the right, 120 hours in the order from the lower left to the right, 168 hours in the order from the lower left to the right) on the 15 mol% PNaAMPS disk with a crosslinking density. Morphology of mouse ES cell colonies on a P (NaAMPS-DMAAm) disk having a crosslink density of 4 mol% and an Fa value of 0.1 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaAMPS-DMAAm) disk with a crosslink density of 4 mol% and an Fa value of 0.3 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaAMPS-DMAAm) disk with a crosslink density of 4 mol% and an Fa value of 0.6 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaAMPS-DMAAm) disk with a crosslink density of 4 mol% and an Fa value of 0.8 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). It is a photographic diagram showing the morphology of mouse ES cell colonies in a 2 mol% PNaSS disk having a crosslinking density (cultured 24 hours, 48 hours, 72 hours in order from the upper left to the right, and cultured in 96 hours, 120 hours in the order from the lower left to the right). It is a photographic diagram showing the morphology of mouse ES cell colonies (48 hours, 72 hours, 96 hours in order from the upper left to the right, 120 hours in the order from the lower left to the right, 168 hours in the order from the lower left to the right) in the 4 mol% PNaSS disk with a crosslinking density. It is a photographic diagram showing the morphology of mouse ES cell colonies (48 hours, 72 hours, 96 hours in order from the upper left to the right, 120 hours in the order from the lower left to the right, 168 hours in the order from the lower left to the right) on the 15 mol% PNaSS disk with a crosslinking density. It is a photograph showing the morphology of mouse ES cell colonies in a PDMAAm disk having a crosslink density of 1 mol% (cultured for 48 hours and 72 hours in order from the upper left to the right, and cultured for 96 hours and 120 hours in the order from the lower left to the right). It is a photograph figure which shows the form of a mouse ES cell colony in a 2 mol% PDMAAm disk with crosslink density (72 hours, 120 hours of culture in order from left to right). Morphology of mouse ES cell colonies on a P (NaSS-DMAAm) disk having a crosslink density of 4 mol% and Fb value of 0.05 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaSS-DMAAm) disk having a crosslink density of 4 mol% and an Fb value of 0.1 (cultured 48 hours, 72 hours, 96 hours in order from upper left to right, sequentially from lower left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaSS-DMAAm) disk having a crosslink density of 4 mol% and an Fb value of 0.2 (48 hours, 72 hours, 96 hours, culture from left to right, sequentially from bottom left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). Morphology of mouse ES cell colonies on a P (NaSS-DMAAm) disk with a crosslink density of 4 mol% and an Fb value of 0.5 (cultured 48 hours, 72 hours, 96 hours, left lower to right sequentially from upper left to right) It is a photograph figure which shows 120 hours of culture | cultivation (168 hours). It is the figure which showed the elastic modulus and swelling degree of each disk. It is the figure which showed the relationship between the crosslinking density in a PNaAMPS disc, and the number of ES cell colonies after 96-hour culture | cultivation. It is a photograph figure which shows the form (120-hour culture | cultivation) of the mouse | mouth ES cell in a 5 wt% PVA disc. It is a photograph figure which shows the form (120-hour culture | cultivation) of the mouse | mouth ES cell in a 10 wt% PVA disc. It is a photograph figure which shows the form (24 hours culture | cultivation) of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a photograph figure which shows the form (culture 48 hours) of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a photograph figure which shows the form (culture 72 hours) of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a photograph figure which shows the form (culture 96 hours) of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a photograph figure which shows the form (120-hour culture | cultivation) of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a photograph figure which shows the state (120-hour culture | cultivation) which mouse | mouth ES cells in a crosslinking density 0.5 mol% PNaAMPS disc stained ALP. It is a photograph figure which shows the state (120-hour culture | cultivation) which carried out the ALP dyeing | staining of the mouse | mouth ES cell in a crosslinking density 4 mol% PNaAMPS disc. It is a photograph figure which shows the state (120-hour culture | cultivation) which carried out the ALP dyeing | staining of the mouse | mouth ES cell in a crosslinking density 10 mol% PNaAMPS disc. It is a photograph figure which shows the state (120-hour culture | cultivation) which mouse | mouth ES cells in a crosslinking density 15 mol% PNaAMPS disc stained. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in a P (NaAMPS-DMAAm) disk whose crosslink density is 4 mol% and Fa value is 0.5. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in the P (NaAMPS-DMAAm) disk whose crosslink density is 4 mol% and Fa value is 0.6. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in a P (NaAMPS-DMAAm) disk whose crosslink density is 4 mol% and Fa value is 0.8. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in the P (NaSS-DMAAm) disk whose crosslink density is 4 mol% and Fb value is 0.2. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in a P (NaSS-DMAAm) disk whose crosslink density is 4 mol% and Fb value is 0.5. It is a photograph figure which shows the state which carried out the ALP dyeing | staining of the mouse | mouth ES cell in the PS disc which is a comparative example. It is a figure which shows the ratio of the colony which exhibited ALP positive among the mouse | mouth ES cell colonies on each disk. It is a figure which shows the expression level of the undifferentiation marker gene of the mouse | mouth ES cell on each disk. It is a figure which shows the expression level of the ectodermal differentiation marker gene of the mouse | mouth ES cell on each disk. It is a figure which shows the expression level of the endoderm type | system | group differentiation marker gene of the mouse | mouth ES cell on each disk. It is a figure which shows the expression level of the mesodermal differentiation marker gene of the mouse | mouth ES cell on each disk. It is a graph which shows the average fluorescence value of the anti- SSEA-1 antibody couple | bonded with the surface of the mouse | mouth ES cell on each disk. In the figure, the black bar represents the average fluorescence value in ES cells cultured in a medium containing LIF, and the hatched bar represents the average fluorescence value in ES cells cultured in a medium not containing LIF.

  Hereinafter, the scaffold material according to the present invention will be described in detail. The scaffold material according to the present invention is made of a hydrogel, and is a material capable of culturing undifferentiated cells such as ES cells, adult stem cells, and iPS cells in an undifferentiated state.

  The “undifferentiated cell” in the present invention means an animal-derived cell having multipotency such as pluripotency and pluripotency. Examples of undifferentiated cells having pluripotency include ES cells and iPS cells, and examples of pluripotent cells include AS cells. Moreover, although it will not specifically limit if it is an animal origin, The origin of a mammal is preferable, the origin of a primate is more preferable, and the origin of a human is further more preferable. Here, the “undifferentiated state” means a state in which a colony of undifferentiated cells is present visually. For example, a state exhibiting an alkaline phosphatase (ALP) staining reaction described later or a gene unique to undifferentiated cells Or the state which expresses an antigen. That is, whether or not the state is undifferentiated can be determined by a method that can be normally selected by those skilled in the art.

  In the present invention, “scaffold material” means “scaffold base material”, “scaffold”, “scaffold”, “culture (for) substrate”, “culture (for) base material”, “culture (for) carrier”. Alternatively, it can be used interchangeably with a “fixing device”.

  The polymer in the present invention may be a so-called hydrogel having a feature that the polymer takes a network structure by chemical bonds and has a large amount of water in the network, but preferably 2-acrylamide- Hydrogel containing 2-methylpropanesulfonic acid (AMPS), p-styrenesulfonic acid (SS) or a salt in which a metal or base is added to the sulfonic acid group as a monomer unit, N, N-dimethylacrylamide (DMAAm) It is a hydrogel selected from the group consisting of a hydrogel containing monomer units, a hydrogel containing vinyl alcohol as monomer units, or a hydrogel containing two or more monomer units.

  One of the preferred hydrogels in the present invention is a hydrogel containing 2-acrylamido-2-methylpropanesulfonic acid (AMPS) or a salt obtained by adding a metal or a base to the sulfonic acid group as a monomer unit. Examples of such gels include poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel, poly (2-acrylamido-2-methylpropanesulfonic acid alkali metal salt) gel, and hydrogels thereof. A hydrogel having a structure in which the second polymer penetrates into the crosslinked network structure of the first polymer can be mentioned, and sodium 2-acrylamido-2-methylpropanesulfonate (NaAMPS) is used as a monomer unit. The poly (2-acrylamido-2-methylpropanesulfonate sodium) (PNaAMPS) gel is more preferable.

Here, NaAMPS is a negatively charged monomer that can be represented by the following structural formula I (Chemical formula 1), and PNaAMPS is a negatively charged polymer that can be represented by the following structural formula II (Chemical formula 2). It is.

  Another preferred hydrogel in the present invention is hydrogel containing p-styrenesulfonic acid (SS) or a salt in which a metal or a base is added to the sulfonic acid group as a monomer unit. Examples of such a gel include poly (p-styrene sulfonic acid) (PSS) gel, poly (p-styrene sulfonic acid alkali metal salt) gel, and these hydrogels are the first polymer and have a crosslinked network. Examples thereof include a hydrogel having a structure in which a second polymer has penetrated into the structure, but preferably contains sodium p-styrenesulfonate (NaSS) as a monomer unit, and poly (sodium p-styrenesulfonate). ) (PNaSS) gel is more preferred.

Here, NaSS is a negatively charged monomer that can be represented by the following structural formula III (Chemical formula 3), and PNaSS has a negative charge that can be represented by the following structural formula IV (Chemical formula 4). It is a polymer.

  Another preferred hydrogel in the present invention is a hydrogel containing N, N-dimethylacrylamide (DMAAm) as a monomer unit. Examples of such gels include poly (N, N-dimethylacrylamide) (PDMAAm) gel and hydrogel having a structure in which PDMAAm is the first polymer and the second polymer penetrates into the crosslinked network structure. PDMAAm is preferable.

Here, DMAAm is a neutral monomer that can be represented by the following structural formula V (Chemical formula 5), and PDMAAm is a neutral polymer that can be represented by the following structural formula VI (Chemical formula 6).

  PNaAMPS gel, PNaSS and PDMAAm can be prepared by well-known or publicly known methods. For example, Chen Y. et al. M.M. Etc. (Chen YM, Shiraishi N., Satokawa H., Kakugo K., Narita T., Gong JP, Osada Y., Yamamoto K., Ando J., Biomaterials 26: 4588-4596 (2005)) It can be prepared by adding a suitable UV initiator and a crosslinking agent to each monomer solution NaAMPS solution, NaSS solution or DMAAm solution and radical polymerization. These UV initiators and cross-linking agents are not particularly limited as long as a PNaAMPS gel can be prepared. For example, 2-Oxglutaricacid can be used as a UV initiator, and N, N-methylenbis-acrylamide (MBAA) can be used as a cross-linking agent. Can be used. In addition, the conditions for the radical polymerization reaction can be appropriately selected. M.M. The reaction conditions in such methods can be followed. Specifically, the reaction temperature is 20 ° C. to 30 ° C., the reaction time is 6 to 10 hours, the addition amount of the UV initiator is 0.1 mol%, and the addition amount of the crosslinking agent is 2 mol% to 10 mol%. It can be selected appropriately. In performing the radical polymerization reaction, it is preferable to replace the dissolved oxygen in the NaAMPS solution, NaSS solution or DMAAm solution with an inert gas by bubbling using an inert gas.

Another preferred hydrogel in the present invention is a hydrogel containing vinyl alcohol (CH ₂ CH—OH; VA) as a monomer unit, more preferably polyvinyl alcohol (PVA). PVA can be prepared by a method of hydrolyzing (saponifying) polyvinyl acetate obtained by polymerizing vinyl acetate or other known or known methods.

  The hydrogel of the present invention may be a hydrogel containing two or more monomer units. As such a gel, for example, PAMPS, poly (2-acrylamido-2-methylpropanesulfonic acid alkali metal salt), PSS or poly (p-styrenesulfonic acid alkali metal salt) is the first polymer, and Examples thereof include a hydrogel having a structure in which PDMAAm, which is the second polymer, has entered the crosslinked network structure. Such a hydrogel is described in, for example, Japanese Patent Application No. 2006-350526 related to the invention by the present inventors, and the contents thereof are included in the present specification.

  In the scaffold material according to the present invention, the form of the hydrogel to be prepared is preferably a flat or disc-like thin film. For example, a monomer solution is filled between two glass plates with an appropriate interval therebetween to perform radical polymerization. It is possible to prepare a flat or disc-shaped thin film by a method such as Moreover, although the thickness of a thin film can be determined suitably, it is preferable to set it as 0.1 mm-10 mm, it is more preferable to set it as 0.2 mm-5 mm, and it is more preferable to set it as 0.5 mm-3 mm.

  The hydrogel prepared as described above can be directly used for culturing undifferentiated cells such as human ES cells, human AS cells, and iPS cells, but in a buffer solution or cell culture medium suitable for culturing undifferentiated cells. It is preferable to swell the hydrogel therein and exchange the solvent in the gel. Examples of suitable buffers include 4-(-2-hydroxyethyl) -piperazin-1-ethanesulfonic acid (HEPES) buffer, fetal bovine serum (FBS), gelatin aqueous solution, PBS, and the like used in the examples. Can do. Moreover, a suitable kind and composition of a cell culture solution can be suitably selected according to the kind of undifferentiated cell.

  A known composition medium according to the exact composition of each component may be used, and examples of such a medium include MEM (Eagle's Minimum Essential Medium) and DMEM (Dulbecco's Modified Eagle's Medium). ), IMDM (Iscove's Modified Dulbecco's Medium), RPMI 1640, AIM-V, ADC, LPM, Ham's F10, Ham's F12, DCCM1, DCCM2, BGJ, BME (Basal Medium Eg G, GME) s Modified Eagle's Medium), L-15 (Leibovitz-15), McCoy5A, M199, Fisher, Schneider, etc. be able to. In addition, if necessary, medium components of known constitution, known balanced salt composition components such as Earl balanced salt solution, Hanks balanced salt solution, Dulbecco PBS, spinner salt solution, various cell growth factors, antibiotics, vitamins , Hormones, pH adjusters, serum, other biological components, and the like can be added.

  In culturing undifferentiated cells, it is preferable to sterilize the hydrogel by UV sterilization or autoclave sterilization.

Cultivation of undifferentiated cells on the hydrogel prepared as described above can be performed by adding a cell suspension containing an appropriate number of undifferentiated cells to the hydrogel placed in an appropriate container. For example, it can be performed by seeding 1 × 10 ⁴ to 1 × 10 ⁶ undifferentiated cells per hydrogel unit area (cm ² ) and placing the hydrogel under standard undifferentiated cell culture conditions. . For example, the culture conditions can be appropriately selected from a culture temperature of 20 ° C. to 40 ° C., a 1% to 10% CO ₂ atmosphere, and a culture time of 24 hours to 168 hours.

  In the present invention, the determination as to whether or not the cell is in an undifferentiated state is performed by observing cell morphology or by alkaline phosphatase (ALP) staining. In the cell morphology observation, the morphology of the cells on the culture disk is observed using a microscope with an appropriate magnification to confirm the collapse of colonies and the presence of differentiated cells. In addition, the ALP staining method adds a reaction solution containing a phosphate ester salt and a diazonium salt as substrates to cells on a culture disk, whereby the phosphate ester salt is hydrolyzed by alkaline phosphatase present in the cell membrane, A coupling reaction with a diazonium salt produces an azo dye, which precipitates at the ALP active site. By counting the number of colonies stained in this way, it becomes possible to quantify the ALP activity of the cells, thereby quantifying the degree of undifferentiation of the cells.

  ALP staining may be performed by appropriately preparing an ALP staining solution, or using a commercially available kit. Examples of such commercially available kits include Leukocyte Alkaline Phosphatase Kit (Sigma), New Fuchsin Substrat System (DAKO), StemTAG Alkaline Phosphate Satin Kit Can be mentioned.

  The determination of whether or not the cell is in an undifferentiated state may be made by quantifying the expression level of a cell differentiation marker gene including an undifferentiated marker gene using RT-PCR (Reverse Transcription Polymerase Chain Reaction) method. Good.

  RT-PCR can be performed according to the description of a normal experiment, and examples of such an experiment can include Sambrook et al. (Molecular Cloning, A laboratory manual, 2001 edition, Cold Spring Harbor Laboratory Press). .

RT-PCR may be performed after extracting total RNA, or so-called real-time RT-PCR may be performed without extracting total RNA. In the former case, for RNA extraction, for example, RNeasy Mini Kit (Qiagen), QIAamp RNA kit (Qiagen), Concert Plant RNA Regent (Invitrogen), QuickPrep RT-PCR methods include, for example, Expand High Fidelity ^PLUS PCR System (Roche), Pyrobest (Takara Bio), ThermoScript RT-PCR System, PLATIUM Taq DNA Polymer G , ABI Prism 7000, 7900 (both are ABI), Sup rscript III First Strand Synthesis System (Invitrogen, Inc.) can be used. On the other hand, in the latter case, for example, Smart Cycler System II (Cepheid), TaqMan Gene Expression Cells-to-CT Kit (ABI), CellAmp Direct RNA Prep Kit For One RT RT-PCR (Tabio) -Time RT-PCR System (Molecular Probes) etc. can be used.

  Further, the determination of whether or not the cell is in an undifferentiated state is carried out by determining the expression of an undifferentiated marker SSEA-1 antigen in embryonic stem cells by staining with an anti-SSEA-1 antibody (Thomson et al., Science, 282 (114), 1998). You may perform by confirming using flow cytometry (Flow cytometry; FCM).

  The flow cytometry can be performed using a flow cytometer or a flow cytometry system, and examples thereof include various flow cytometers and flow cytometry systems such as Becton Dickinson and Beckman Coulter.

  Hereinafter, the scaffold material according to the present invention will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1>
(1) Preparation of PNaAMPS gel Sodium 2-acrylamido-2-methyl-1-propanesulfonate (NaAMPS), 0.1 mol% 2-Oxglutaric acid as a UV initiator, and 4 mol% N, N as a crosslinking agent -20 mL of an aqueous solution containing methylenebisacrylamide (MBAA) was prepared, and dissolved oxygen was replaced with nitrogen by nitrogen bubbling. Six types of monomer concentrations (crosslink density) of PNaAMPS were prepared: 0.5 mol%, 1 mol%, 2 mol%, 4 mol%, 10 mol%, and 15 mol%.

  Each monomer solution after nitrogen substitution was poured into a mold formed by sandwiching a 1.5 mm silicon spacer between two 10 × 10 mm square glass substrates, and then polymerized at room temperature for 6 hours to obtain a crosslinking density. Poly (2-acrylamido-2-methyl-1-propanesulfonate sodium) (PNaAMPS) gels were obtained which were 0.5 mol%, 1 mol%, 2 mol%, 4 mol%, 10 mol% and 15 mol%, respectively.

Each hydrogel was taken out from the glass substrate and swollen for 1 week by exchanging 1 L of distilled water every day, and then the hydrogel was 5 mM 4-(-2 containing 15.5 mM NaHCO ₃ and 140 mM NaCl. -Hydroxyethyl)-Piperazine-1-Ethersulfonic acid (HEPES) buffer solution (pH 7.4) was immersed for one week to perform solvent exchange of the hydrogel.

(2) Preparation of P (NaAMPS-DMAAm) gel Fa (molar ratio) = [NaAMPS monomer concentration (crosslinking density)] / [N, N-dimethylacrylamide (DMAAm) monomer concentration (crosslinking density) + NaAMPS monomer concentration (crosslinking density) )], And this Fa value is 0.1, 0.2, 0.3, 0.5, 0.6 and 0.8, respectively, and the crosslink density is 4 mol%. Sodium 2-acrylamido-2-methyl-1-propanesulfonate)-(N, N-dimethylacrylamide)} {P (NaAMPS-DMAAm)} gel was prepared.

  Specifically, UV (wavelength 365 nm) was applied to an aqueous solution containing 1 mol / L NaAMPS, 4 mol% MBAA with respect to NaAMPS and 0.1 mol% α-ketoglutaric acid with respect to NaAMPS for 6 hours, and the degree of crosslinking was 4 mol%. PNaAMPS was obtained.

  1 mol / L NaAMPS and DMAAm monomer mixed solution according to Fa value, 4 mol% MBAA with respect to NaAMPS and DMAAm monomer mixed solution, and 0.1 mol% α-ketoglutaric acid with respect to NaAMPS and DMAAm monomer mixed solution After replacing the dissolved oxygen with nitrogen by nitrogen bubbling, it was poured into a mold formed by sandwiching a 1.5 mm silicon spacer between two 10 × 10 mm square glass substrates, sealed, and then UV ( (Wavelength 365 nm) for 6 hours, Fa values of 0.1, 0.2, 0.3, 0.5, 0.6 and 0.8, respectively, and P ( NaAMPS-DMAAm) gel was obtained. Solvent exchange of the obtained P (NaAMPS-DMAAm) gel was performed in the same manner as in Example (1).

(3) Preparation of hydrogel disk Each hydrogel of Examples (1) and (2) after solvent exchange was transferred to a petri dish, and autoclaved at 121 ° C. for 20 minutes. 3 mm hydrogel disc {crosslink density 0.5 mol% PNaAMPS disc, 1 mol% PNaAMPS disc, 2 mol% PNaAMPS disc, 4 mol% PNaAMPS disc, 10 mol% PNaAMPS disc, 15 mol% PNaAMPS disc, and crosslink density 4 mol% each P (NaAMPS-DMAAm) disk having a Fa value of 0.1, 0.2 P (NaAMPS-DMAAm) disk, 0.3 P (NaAMPS-DMAAm) disk, 0.5 P (N AMPS-DMAAm) disk, 0.6 in P (NaAMPS-DMAAm) disks and 0.8 P a (NaAMPS-DMAAm) disc} was prepared.

(4) Cultivation of mouse ES cells on hydrogel disk Each of the disks prepared in Example (3) was placed in a 24-well polystyrene (PS) dish, and 20% fetal bovine serum was used as an undifferentiated cell culture solution from above. After adding 0.5 mL / well of DMEM (IBL) containing (fetal bovine serum; FBS) and removing this solution after 24 hours, 1.25 × 10 ⁴ cells / mL of MGZ5 mouse embryonic stem cells (hereinafter referred to as “mouse ES”). Cell).) 1 mL / well of suspension (DMEM with 20% FBS) was added. The 24-well PS dish was placed in an incubator at 37 ° C. and 5% CO ₂ atmosphere to culture mouse ES cells.

<Example 2>
(1) Preparation of PNaSS gel disk A disk having a diameter of 15 mm and a thickness of 1 to 3 mm made of poly (sodium p-styrenesulfonate) (PNaSS) gel having crosslink densities of 2 mol%, 4 mol%, 10 mol% and 15 mol%, respectively. Prepared according to the method described in Examples 1 (1) and (3).

(2) Preparation of P (NaSS-DMAAm) gel disk Fb (molar ratio) = [p-sodium styrenesulfonate (NaSS) monomer concentration (crosslinking density)] / [DMAAm monomer concentration (crosslinking density) + NaSS monomer concentration (crosslinking) Density)], which has an Fb value of 0.05, 0.1, 0.2, and 0.5, respectively, and a crosslink density of 4 mol%, poly {(sodium p-styrenesulfonate) -Hydrogel disc made of-(N ', N-dimethylacrylamide)} {P (NaSS-DMAAm)} gel having a diameter of 15 mm and a thickness of 1 to 3 mm {all crosslink density is 4 mol% and Fb value is 0 respectively. .05 P (NaSS-DMAAm) disk, 0.1 P (NaSS-DMAAm) disk, 0.2 P (NaSS-DM The Am) disks and 0.5 of P (NaSS-DMAAm) disk}, was prepared according to the method described in Example 1 (2) and (3).

(3) Cultivation of mouse ES cells on hydrogel disk Each PNaSS disk and P (NaSS-DMAAm) disk prepared in Comparative Examples (1) and (2) were prepared in the same manner as in Example 1 (3). The cells were sterilized by autoclaving and placed in a 24-well PS dish under the conditions described in Example 1 (4) to culture mouse ES cells.

<Example 3>
(1) Preparation of PDMAAm gel disk A disk having a diameter of 15 mm and a thickness of 1 to 3 mm made of a poly (N, N-dimethylacrylamide) (PDMAAm) gel having a crosslinking density of 1 mol% and 2 mol%, respectively, was prepared in Example 1 (1). ) And (3).

(2) Culture of mouse ES cells on hydrogel disk Each PDMAAm disk prepared in Example (1) was autoclaved in the same manner as in Example 1 (3) and described in Example 1 (4). Under these conditions, each mouse ES cell was cultured in a 24-well PS dish.

<Example 4>
(1) Preparation of PVA gel disk PVA powder (polymerization degree 2,000, molecular weight about 90,000) mixed solvent of dimethyl sulfoxide (DMSO) and water (DMSO and water so that the molecular weight is about 5% and 10% by weight). Each solution dissolved at 90 ° C. in a weight ratio of 3: 1) was poured into a glass mold in the same manner as in (2) of Example 1, frozen at −40 ° C. for 16 hours, and then thawed at room temperature. A gel was formed. After each gel was removed from the glass substrate and allowed to swell for 1 week by changing 1 L of distilled water daily, the gel was washed with 5 mM 4-(-2 containing 15.5 mM NaHCO ₃ and 140 mM NaCl. -Hydroxyethyl) -piperazin-1-ethanesulfonic acid (HEPES) buffer (pH 7.4) was immersed in the solution for one week to perform solvent exchange to prepare 5% PVA gel disc and 10% PVA gel disc.

(2) Culture of mouse ES cells on hydrogel disk Each PVA gel disk prepared in Example (1) was immersed in 75% ethanol and sterilized overnight using a UV lamp. Under the conditions described in Example 1 (4), each mouse ES cell was cultured in a 24-well PS dish.

<Comparative example>
A PS disk was prepared by adding 0.4 mL / well of 0.1% gelatin directly from the top of a 24-well PS dish and placing it in a 37 ° C., 5% CO ₂ atmosphere incubator for 30 minutes for gelatin coating. Mouse ES cells were cultured under the conditions described in Example 1 (4) and used as comparative examples.

<Test Example 1> Morphological observation of mouse ES cell colonies For each hydrogel disk of Examples 1 to 4 and PS disks prepared as comparative examples, the morphology of mouse ES cell colonies on the disk was measured using a phase contrast microscope. Observed in time series, the following results (1) to (6) were obtained.

(1) Morphology of mouse ES cell colonies on PNaAMPS discs In a PNaAMPS disc with a crosslink density of 0.5 mol%, mouse ES cell colonies grew well, and colonies with clear outlines were confirmed after 120 hours of culture. (FIG. 1). On a 2 mol% PNaAMPS disk with a crosslink density, mouse ES cell colonies having a diameter of about 10 μm were formed after 48 hours of culture, and grown to a diameter of about 15 to 35 μm after 168 hours of culture. In addition, on the 2 mol% PNaAMPS disk having a crosslinking density, although differentiated cells were slightly observed after 96 hours of culture, only a small number of differentiated cells were observed thereafter (FIG. 2). On the 15 mol% PNaAMPS disk with a crosslinking density of 15 μm, a mouse ES cell colony having a diameter of about 10 μm was formed after 48 hours of culture, and it grew to a diameter of about 25 to 55 μm after 168 hours of culture. Differentiated cells were observed, and the collapse of the outline of the mouse ES cell colony was confirmed by culturing for 120 hours. After further culturing for 168 hours, the cross-linking density was differentiated compared to that on the 2 mol% PNaAMPS disc. Many cells were observed (FIG. 3).

(2) Morphology of mouse ES cell colonies on P (NaAMPS-DMAAm) discs On P (NaAMPS-DMAAm) discs with a crosslink density of 4 mol% and a Fa value of 0.1, mouse ES cell colonies are good It grew to a diameter of about 50 μm after 168 hours of culture. However, the number of observed mouse ES cell colonies tended to decrease with time (FIG. 4). In addition, mouse ES cell colonies grow well even on P (NaAMPS-DMAAm) disks having a crosslink density of 4 mol% and an Fa value of 0.3, but the outline of the mouse ES cell colonies is broken after 120 hours of culture. Beginning to be confirmed, mouse ES cell colonies were not observed after 168 hours (FIG. 5). Even in the case of P (NaAMPS-DMAAm) disk having a crosslink density of 4 mol% and Fa value of 0.6 and 0.8 P (NaAMPS-DMAAm) disk, the crosslink density was 4 mol%. A tendency similar to that on a P (NaAMPS-DMAAm) disk having an Fa value of 0.3 was shown (FIGS. 6 and 7).

(3) Morphology of mouse ES cell colonies on PNaSS disc On the 2 mol% PNaSS disc having a crosslinking density, it was confirmed that the outline of mouse ES cell colonies was maintained until after 72 hours of culture. Thereafter, although this collapse of the outline was confirmed, mouse cell colonies could be observed even after 120 hours of culture (FIG. 8). On the 4 mol% PNaSS disk with a crosslinking density of 4 to 20 μm, mouse ES cell colonies having a diameter of about 10 to 20 μm were formed after 48 hours, and the mouse ES cell colonies had grown to a diameter of about 25 to 55 μm after 96 hours. . However, a slight collapse was observed in the outline of the colony after 96 hours of culture (FIG. 9). The same tendency was observed when the crosslink density was 15 mol% PNaSS disk (FIG. 10).

(4) Morphology of mouse ES cell colonies on PDMAAm disc On the 1 mol% PDMAAm disc and 2 mol% PDMAAm disc, respectively, the growth of mouse ES cell colonies is observed regardless of the crosslink density of PDMAAm. The growth rate began to slow after 72 hours of culture and almost stopped after 120 hours of culture (FIGS. 11 and 12).

(5) Morphology of mouse ES cell colonies on P (NaSS-DMAAm) discs In P (NaSS-DMAAm) discs with a crosslink density of 4 mol% and Fb value of 0.05, mouse ES cell colonies are cultured for a period of time. And grew to a diameter of about 50 μm after 168 hours of culture. However, the number of observed mouse ES cell colonies tended to decrease with time (FIG. 13). A P (NaSS-DMAAm) disk having a crosslink density of 4 mol% and an Fb value of 0.1 showed a similar tendency (FIG. 14). On the other hand, on the P (NaSS-DMAAm) disk and the P (NaSS-DMAAm) disk each having a crosslink density of 4 mol% and an Fb value of 0.2, mouse ES cell colonies are not cultured at the time of culture. Although it grows with it, collapse of the outline of the colony was confirmed after 120-hour culture (FIGS. 15 and 16).

  The elastic modulus and the degree of swelling of PNaAMPS disks having a crosslink density of 0.5 mol%, 2 mol%, 4 mol%, 10 mol% and 15 mol%, respectively, and PNaSS disks having a crosslink density of 2 mol%, 4 mol%, 10 mol% and 15 mol%, respectively. The table shown in FIG. 17 is a graph showing the relationship between the crosslinking density in the PNaAMPS disc and the number of ES cell colonies after 96 hours of culture.

(6) Morphology of mouse ES cell colonies on PVA disc Growth of mouse ES cell colonies was observed until 5 hours in both 5 wt% PVA gel disc and 10 wt% PVA gel disc (FIGS. 19 and 20). .

(7) Morphology of mouse ES colonies on PS disk The results (photos) of the morphology of mouse ES cell colonies on a PS disk as a comparative example were observed in time series using a phase contrast microscope (photos). 25. Cells extended on the PS disc after 24 hours of culture were observed. These cells proliferated with the culture time, and after 72 hours of cultivation, a large amount of expanded cells and a small amount of small round cells were observed. Further, after 96 hours of culturing, a state in which small round cells overlapped with the extended cells was observed, and after 120 hours of culturing, cells in such a state increased in large quantities. On the other hand, unlike the case of using the hydrogel, colony formation by cells could not be confirmed on the PS disk during the culture process. These results inferred that many of the cells cultured on PS disks were differentiated cells.

  From the above, according to the results of microscopic observation of cell morphology, it can be seen that on any hydrogel, mouse ES cells form colonies and proliferate in an undifferentiated state. Further, when a PNaAMPS disk and a PNaSS disk having the same crosslinking density are compared, it can be seen that mouse ES cell colonies grow on the PNaAMPS disk and the number of differentiated cells is smaller than that on the PNaSS disk. Also, when comparing P (NaAMPS-DMAAm) discs with PDMAAm discs, the number of mouse ES cell colonies on the PDMAAm discs tends to decrease over time, but P (NaAMPS-DMAAm) discs are used. In this case, the Fa value does not reduce the number of mouse ES cell colonies grown as compared with the case of using the PDMAAm disc, and it can be seen that the morphology is maintained. Furthermore, when PNAAMPS discs with different crosslink densities were compared with each other, the number of mouse ES cell colonies that grew on a PnaAMPS disc with a low crosslink density, ie, a low modulus of elasticity, was higher than that of a PNaAMPS disc with a high modulus of elasticity. You can see that

<Test Example 2> Mouse ES cell undifferentiation confirmation test (ALP staining)
(2) ALP staining PNAAMPS discs having a crosslink density of 0.5, 4, 10, and 15 mol% prepared in Example 1 (3) and a crosslink density of 4 mol%, respectively, and an Fa value of 0.5, P (NaAMPS-DMAAm) disks with 0.6 and 0.8, and P (NaSS) having a crosslink density of 4 mol% and Fb values of 0.2 and 0.5, respectively, prepared in Example 2 (2). -DMAAm) disk and mouse ES cells were cultured on each disk of the PS disk prepared in Comparative Example for 120 hours, washed with PBS, and the cells in the wells were protocol of Alkaline Phosphatase Detection Kit from Millipore (CHEMICON) According to ALP staining. The results (photographs) are shown in FIGS. Moreover, the graph which showed the ratio of the colony which exhibited ALP positive among the mouse | mouth ES cell colonies on each disk is shown in FIG.

  Based on the above, in mouse ES cells cultured for 120 hours on 0.5 mol% PNaAMPS disc, 4 mol% PNaAMPS disc, 10 mol% PNaAMPS disc, and 15 mol% PNaAMPS disc, respectively, 90% or more of the colonies are ALP positive. I understand that. In addition, on the PNaAMPS disk having a crosslink density of 0.5 mol% and the P (NaAMPS-DMAAm) disk having a crosslink density of 4 mol% and a Fa value of 0.5, the percentage of mouse ES cell colonies exhibiting ALP positivity is the highest. I understand that it is expensive.

<Test Example 3> Quantification of cell differentiation marker gene expression by RT-PCR method The crosslink densities prepared in Example 1 (3) were 0.5 mol% PNaAMPS disc, 1 mol% PNaAMPS disc, 2 mol% PNaAMPS disc, 4 mol%, respectively. From mouse ES cells cultured on PNaAMPS disc, 10 mol% PNaAMPS disc and 15 mol% PNaAMPS disc for 120 hours, using TaqMan GAPDH Control Reagents (ABI) primers and Real Time One Step RNA PCR Kit (Takara Bio) A template sample was prepared. Subsequently, using the expression level of each gene of mouse ES cells on the PS disk as a control, Nanog, Oct3 / 4, SOX2 and DPPA5 / ESG1 (hereinafter sometimes referred to as “DPPA5”), which are undifferentiated marker genes, and the like. In order to examine the expression level at the mRNA level of Nestin and Otx2 which are germ layer differentiation marker genes, GATA-4, SOX17 and NHF-3βFoxA2 which are endoderm differentiation marker genes and Brachyury which is a mesodermal differentiation marker gene, In the presence of SYBR Green I (Takara Bio Inc.), which is an intercalator, real-time One Step RT-PCR was performed using Smart Cycler System II (Cepheid Corp.). The results are shown in FIGS.

  The expression level of Nanog, an undifferentiated marker gene, is large in mouse ES cells cultured on PNaAMPS discs regardless of crosslink density, and the expression level of SOX2 is on PNaAMPS discs other than 0.5 mol% PNaAMPS discs. It can be seen that there are many in cultured mouse ES cells. In addition, the expression level of Oct3 / 4 and DPPA5 is the highest in mouse ES cells cultured on 0.5 mol% PNaAMPS, but it is found that there is almost no difference from that cultured on PS disks (FIG. 37). ).

  Regarding the expression level of nestin and Otx2 which are ectoderm differentiation marker genes, the expression level of Nestin in mouse ES cells cultured on PNaAMPS discs is small regardless of crosslink density, and the expression level of Otx2 is on PS discs. It can be seen that the expression is equivalent to or higher than that in the culture (FIG. 38).

  The expression levels of the endoderm differentiation marker genes GATA-4, SOX17, and NHF-3βFoxA2 are all high in mouse ES cells cultured on PS disks, and low in mouse ES cells cultured on PNaAMPS disks. (FIG. 39).

  It can be seen that the expression level of Brachyury, a mesoderm differentiation marker gene, is high in mouse ES cells cultured on PS discs and low in mouse ES cells cultured on PNaAMPS discs (FIG. 40).

<Test Example 4> Confirmation of undifferentiated state using flow cytometry According to the method described in Examples 1 to 4, 0.5 mol% PNaAMPS disc, 2 mol% PNaAMPS disc, 10 mol% PNaAMPS disc, 0.5 mol% Poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) disk, 2 mol% PAMPS disk, 2 mol% PNaSS disk, 4 mol% PNaSS disk, 15 mol% PNaSS disk, 5 wt% PVA disk, 10 wt% PVA disk, 1 mol% PDMAAm discs, 8 mol% PDMAAm discs and 15 mol% PDMAAm discs were prepared.

  For each of the above disks, the culture shown in Test Example 1 does not contain a medium containing chicken leukemia inhibitory factor (LIF), which is known to have a function of inhibiting the differentiation of mouse ES cells, and this. Each was performed using a medium.

  1 mL of PBS solution was added to each disk after culture to wash ES cells, and then 1 mL of 5 mM EDTA / PBS solution was further added. After standing at room temperature for 15 minutes, the cells were peeled off from the gel with a cell scraper to prepare an EDTA / PBS solution containing the recovered cells. 5 mL of this solution was transferred to a polystyrene round bottom tube and centrifuged at 1300 rpm and 4 ° C. for 5 minutes to collect cells. To this, 5 μL of mouse anti-SSEA-1 antibody (BD Pharmingen) labeled with FITC (fluorescein isothiocyanate) was added, and the cells were allowed to stand on ice for 1 hour in the dark. Thereafter, 1 mL of a PBS solution containing 1% bovine serum albumin (BSA) was added, followed by washing twice by centrifugation at 1300 rpm and 4 ° C. for 5 minutes. 200 μL of PBS solution containing 1% BSA was added to the washed cells, and the fluorescence value of FITC-labeled mouse anti-SSEA-1 antibody bound to the cell surface was measured with a flow cytometer (Becton Dickinson). The control prepared what processed the cell cultured on the PS disk of a comparative example like the above. The average value of the previous fluorescence value is shown in FIG.

  Compared with the mean fluorescence value for ES cells on PS disk as a control, the mean fluorescence values for ES cells on hydrogel discs according to the present invention are both high, especially for ES cells on PVA disc and PDMAAm disc. The average fluorescence value was 10 to 20 times the average fluorescence value of the control. These results indicate that ES cells cultured on the hydrogel disk of the present invention maintain high undifferentiation.

  In addition, when LIF is not present in the medium, the average fluorescence value of ES cells cultured on the negatively charged PNaAMPS disk and PNaSS disk is higher than the average fluorescence value when LIF is present in the medium. The average fluorescence values of ES cells cultured on PVA discs and PDMAAm discs with neutral charge were almost the same when LIF was present in the medium and when LIF was not present.

  From the above examples and test examples, mouse ES cells cultured on PNaAMPS discs tend to remain undifferentiated, but mouse ES cells cultured on PS discs differentiate into endoderm or mesodermal cells. It can be seen that there is a trend.

  As described above, oxygen and nutrients can be sufficiently and efficiently supplied to undifferentiated cells without causing problems such as virus infection and contact with heterologous cells, and undifferentiated cells can be replenished on the hydrogel. It can be cultured in a differentiated state.

  In addition, the scaffold material which concerns on this invention is not limited to the Example mentioned above, It can change suitably. For example, the scaffold material according to the present invention may be used for culturing differentiated cells or progenitor cells.

Claims (2)

  A scaffold material for culturing undifferentiated cells in an undifferentiated state, the scaffold material comprising a hydrogel.   The hydrogel includes 2-acrylamido-2-methylpropanesulfonic acid, styrenesulfonic acid or a salt obtained by adding a metal or a base to the sulfonic acid group as a monomer unit, and hydrogel includes dimethylacrylamide as a monomer unit. The scaffold material according to claim 1, which is a hydrogel selected from the group consisting of a hydrogel containing vinyl alcohol as a monomer unit or a hydrogel containing two or more kinds of the monomer units.