WO2019163948A1

WO2019163948A1 - Substrate for cell culture and culture method

Info

Publication number: WO2019163948A1
Application number: PCT/JP2019/006806
Authority: WO
Inventors: 祥子西原; 三浦　太一; 祐二松▲崎▼; 徳行湯浅; 正人羽生
Original assignee: 学校法人創価大学; 東京化成工業株式会社
Priority date: 2018-02-23
Filing date: 2019-02-22
Publication date: 2019-08-29
Also published as: JP7373100B2; JPWO2019163948A1

Abstract

The purpose of the present invention is to provide a cell culture method for hPSC that is suitable for industrial cell production and a substrate for cell culture. For example, the purpose of the present invention is to provide a culture method, whereby hPSC can be cultured and proliferated while maintaining the undifferentiated state or while maintaining the pluripotency without using supporting cells (feeder cells) impeding large-scaled cell production, and a culture substrate. Provided is a method for culturing pluripotent stem cells while maintaining the undifferentiated state, said method comprising a step for culturing cells in a medium containing sulfated hyaluronic acid but containing neither supporting cells nor bFGF, characterized in that the sulfated hyaluronic acid has a degree of sulfation of 7% or greater in terms of S content. Also provided is a substrate for cell culture that is coated with sulfated hyaluronic acid having a degree of sulfation of 7% or greater in terms of S content.

Description

Cell culture substrate and culture method

The present invention relates to a cell culture substrate and a culture method in the field of cell culture.

Since the establishment of embryonic stem cells (ES cells) in humans that have the ability to self-replicate indefinitely while maintaining the pluripotency that can differentiate into all cells of an individual, Many studies have been conducted to induce differentiation into major organ cells. In addition, by introducing a specific gene into somatic cells, human induced pluripotent stem cells (iPS cells) having both differentiation pluripotency and self-replication ability like ES cells are created. Studies have been actively conducted to induce differentiation into major organ cells in vitro using iPS cells.

These pluripotent stem cells have the ability to differentiate into cells of various tissues and organs and the ability to proliferate almost infinitely. Therefore, elucidation of the cause of diseases, development of new drugs, cell transplantation therapy, etc. Applications are expected. In particular, human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) are expected to be applied to drug discovery and regenerative medicine. (Non-patent document 1: D. A. Robinton et al., Nature. 2012 Jan 18; 481 (7381): 295-305.).

Culturing and proliferating pluripotent stem cells safely and with good reproducibility is an essential technique for applying these cells. In particular, in the field of regenerative medicine, since stem cells need to be handled in large quantities in an undifferentiated state, a technology that supports the proliferation of pluripotent stem cells and maintains pluripotency using natural and synthetic polymers. (Non-patent document 2: Fan Y., et al., Stem Cell Rev. 2015 Feb; 11 (1): 96-109., Non-patent document 3: Chen YM, et al ., Sci Rep. 2017 Mar 23; 7: 45146.).

Currently, the most commonly used techniques for maintaining hPSCs in an undifferentiated state are on feeder cells (eg, mouse embryonic fibroblasts (MEF), human foreskin fibroblasts, human adipose derived cells, etc.) This is a method of culturing (Non-patent Document 4: LG Villa-Diaz et al., Stem Cells. 2013 Jan; 31 (1): 1-7.). However, culturing hPSCs using feeder cells is time consuming and has a problem of variation from lot to lot of feeder cells, and there is also a concern about contamination of substances from feeder cells.
Alternatively, hPSC is cultured on an extracellular matrix, for example, on Matrigel or Geltrex. An extracellular matrix for cell culture such as matrigel contains, as main components, laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen, and further includes various growth factors, etc. Engelbreth-Holm-Swarm (EHS) It consists of a substrate isolated from mouse sarcoma. These have been reported to support the pluripotency of many hESC strains (Non-patent Document 5: KG Chen, et al., Cell Stem Cell. 2014 Jan 2; 14 (1): 13 -26.), Since the exact composition is not clear and it contains components derived from different organisms, the use of such culture conditions may hinder clinical application of hPSC.
In addition, when MEF or Matrigel is used, there are concerns regarding immunogenic epitopes such as N-glycolylneuraminic acid (Neu5Gc) that are not derived from humans and the transmission of heterologous infectious viruses to humans.

Although it is currently required to produce cells such as hESC and hiPSC on a large scale in a facility that is compatible with GMP, the presence of supporting cells particularly hinders the use of hPSC.
In order to maintain the pluripotency of hPSC, it is necessary to add basic fibroblast growth factor (bFGF) to the medium. However, bFGF is expensive and stably stored for a long period of time. This also hinders the industrial production of hPSCs.
FGF is generally added to a medium in order to stably cultivate stem cells, and is one of highly important components even in a serum-free medium. However, it is known that the stability in the medium is low, and it is a factor that requires a high frequency medium exchange in cell culture. This also hinders the industrial production of hPSC.

Until now, it has been reported that certain sulfated polysaccharides have an action of protecting FGF from degradation, denaturation, inactivation, and the like. For example, Patent Document 1 (WO92 / 13526 pamphlet) discloses that carrageenan stabilizes bFGF. Patent Document 2 (Japanese Patent Laid-Open No. 02-138223) discloses a method for stabilizing FGF or a mutein thereof, which comprises contacting FGF or a mutein thereof with a sulfated glucan in an aqueous medium. . Non-Patent Document 6 (J. Cell. Physiol., 1986, 128, 475-484) describes that heparin or hexuronylhexosaminoglycan sulfate (HHS-4) protects bFGF from inactivation and its physiological activity. Is described.

On the other hand, a method for culturing human cells containing anion-modified hyaluronic acid in the medium has been reported (Patent Document 3: Japanese Patent Application Laid-Open No. 2006-211920). It describes that the growth of human mesenchymal cells is promoted by anion-modified hyaluronic acid.

In addition, a biological tissue filler coated with sulfated hyaluronic acid, which is a sulfated polysaccharide, has been reported (Patent Document 4: Japanese Patent No. 5088864). There, it is described that a biological tissue filling agent coated with sulfated hyaluronic acid having a sulfation degree of 0.6 or 1.0 promotes the proliferation of chondrocytes and the ability to differentiate into cartilage. .
In addition, a culture medium and a culture method that can activate cells using sulfated hyaluronic acid and promote differentiation and maturation into neurons have been reported (Patent Document 5: JP 2009-278873 A). . There, sulfated hyaluronic acid having a degree of sulfation of 0.4 or 1.0 is used. By adding a combination of sulfated hyaluronic acid and FGF-2, cells are activated and nerves are activated. It is described that differentiation / maturation into cells can be promoted.

WO92 / 13526 pamphlet Japanese Patent Laid-Open No. 02-138223 JP 2006-211192 A Japanese Patent No. 5088864 JP 2009-278873 A

An object of the present invention is to provide a new cell culture substrate or culture method capable of improving the culture efficiency in culturing hPSCs or hybridomas producing antibodies. *

As a result of intensive studies, the present inventors have found that, by using sulfated hyaluronic acid having a relatively high degree of sulfidation, the culture efficiency can be improved in the culture of hPSC and the hybridoma that produces antibodies, The present invention has been completed.
In one aspect of the present invention, by using sulfated hyaluronic acid having a relatively high degree of sulfidation, hPSC can be cultured and grown in an undifferentiated state or in a state of maintaining pluripotency without using feeder cells. be able to.
In another aspect of the present invention, by using sulfated hyaluronic acid having a relatively high degree of sulfidation, the amount of antibody production can be increased in the culture of antibody-producing hybridomas.
More specifically, the present invention includes the following cell culture method and cell culture substrate.

(Cell culture method)
One aspect of the present invention is a sulfated hyaluronic acid having a relatively high degree of sulfation (the degree of sulfation is the number of sulfate groups per disaccharide unit of hyaluronic acid (hereinafter referred to as “sulfation degree (number of sulfate groups / 2 disaccharide units)”). By using a sulfated hyaluronic acid that is about 1.3 or more, preferably about 1.5 or more, more preferably about 2.0 or more) without using supporting cells. However, the present invention provides a cell culture method for culturing and growing hPSC in an undifferentiated state or in a state where pluripotency is maintained.
In another aspect, the present invention has a relatively high degree of sulfation (sulfation degree (number of sulfate groups / 2 sugar units) of about 1.3 or more, preferably about 1.5 or more, more preferably about 2.0 or more. (And more preferably, about 3.0 or more) by using sulfated hyaluronic acid, hPSC can be cultured and proliferated in an undifferentiated state or in a state of maintaining pluripotency without using supporting cells and bFGF. A method for cell culture is provided.

(Substrate for cell culture)
In another aspect of the present invention, in the culture of hPSCs that do not use feeder cells, and optionally do not use bFGF in addition to feeder cells, the cells are cultured in a state in which hPSCs are maintained in an undifferentiated state or in a state that maintains multipotency Sulfated hyaluronic acid having a relatively high sulfation degree (sulfation degree (sulfuric acid group / 2 sugar units) of about 1.3 or more, preferably about 1.3 to about 3.0) for growth is A coated cell culture substrate is provided, optionally with an extracellular matrix.
In another aspect, the present invention provides a method for culturing hPSC using the cell culture substrate described above.

In still another aspect of the present invention, a relatively high degree of sulfation (sulfation degree (sulfuric group number / 2 sugar units) is preferably about 1.3 or more, more preferably about 2.0 or more, and still more preferably. Provided is a method for producing an antibody by culturing a hybridoma having an antibody-producing ability using a medium containing about 3.0 or more of sulfated hyaluronic acid.

Specifically, the present invention includes the following aspects.
[1] A cell culture substrate having a surface coated with sulfated hyaluronic acid.
[2] The cell culture substrate according to [1], wherein the sulfated hyaluronic acid is sulfated hyaluronic acid having an S content of about 7% or more (preferably 10% or more).
[3] The cell culture substrate according to the above [1], wherein the sulfated hyaluronic acid is introduced at a ratio of at least about 1.3 or more on average with respect to a disaccharide unit as a structural unit. Wood.
[4] The sulfated hyaluronic acid according to the above [3], wherein the sulfate group is introduced at an average ratio of about 1.3 to about 3.0 with respect to the disaccharide unit which is a structural unit. Cell culture substrate.
[5] The cell culture substrate according to [3], wherein the sulfated hyaluronic acid is introduced at a ratio of 3.0 or more on average to a disaccharide unit as a constituent unit. .
[6] A substrate for culturing pluripotent stem cells, which is used for culturing for maintaining pluripotent stem cells in an undifferentiated state. The cell culture substrate according to 1.
[7] The cell culture substrate according to [6], wherein the pluripotent stem cells are iPS cells.
[8] The cell culture substrate according to any one of the above [1] to [7], wherein the sulfated hyaluronic acid is coated on the surface together with an extracellular matrix, protein or polymer.
[9] The cell culture substrate according to [8] above, wherein the sulfated hyaluronic acid is present in a state of being uniformly dispersed in an extracellular matrix, protein or polymer.
[10] A cell culture instrument comprising the cell culture substrate according to any one of [1] to [9] above.
[11] Using the cell culture substrate according to any one of [1] to [9] above, pluripotent stem cells (preferably ES cells or iPS cells) are cultured, and their differentiation potential is increased. A method for culturing pluripotent stem cells, comprising maintaining the pluripotent stem cells.

[12] A method for culturing pluripotent stem cells (preferably ES cells or iPS cells), comprising the following steps:
A step of culturing cells using a culture medium to which sulfated hyaluronic acid is added under culture conditions not containing feeder cells, wherein the sulfated hyaluronic acid has an S content of about 7% or more (preferably about 10 %, More preferably about 13% or more, and still more preferably about 14% or more).
A culture method comprising:
[13] A method for culturing pluripotent stem cells (preferably ES cells or iPS cells), comprising the following steps:
A step of culturing cells under a culture condition without supporting cells using a medium to which sulfated hyaluronic acid is added, wherein the sulfated hyaluronic acid has a sulfate group with respect to a disaccharide unit as a constituent unit; Introduced at a rate of at least about 1.3 on average (preferably about 2.0 or more, more preferably about 2.5 or more, more preferably about 3.0 or more, even more preferably about 3.5 or more) Being
A culture method comprising:
[14] The culture method according to [12] or [13] above, wherein the culture condition is a culture condition not further containing bFGF.
[15] The culture method according to any one of the above [12] to [14], wherein the culturing step is performed in the presence of an extracellular matrix, protein or polymer.
[16] The culture method according to any one of [12] to [15] above, wherein the pluripotent stem cells are human iPS cells.
[17] A method for producing an antibody by culturing a hybridoma having the ability to produce an antibody, comprising the following steps:
A step of culturing cells in a medium to which sulfated hyaluronic acid is added, wherein the sulfated hyaluronic acid has an S content of 7% or more (preferably 10% or more, more preferably 13% or more, more preferably 14 % Of sulfation degree)
A method comprising the steps of:
[18] A method for producing an antibody by culturing a hybridoma having the ability to produce an antibody, comprising the following steps:
A step of culturing cells in a medium to which sulfated hyaluronic acid is added, wherein the sulfated hyaluronic acid has an average of at least 1.3 sulfate groups (preferably 2 or more) with respect to a disaccharide unit as a constituent unit. 0.0 or more, more preferably 2.5 or more, more preferably 3.0 or more, and still more preferably 3.5 or more).
A method comprising the steps of:

According to the present invention, the culture efficiency can be improved in the culture of hPSC and the hybridoma that produces antibodies.

It is the result of counting the number of colonies after adding 10 μg / ml hyaluronic acid (HA) and highly sulfated hyaluronic acid (HA-HS) without adding feeder cells and bFGF, respectively, and culturing for 3 days. Two types of human iPS cell lines, hiPS # 16 (left) and hiPS # 25 (right), were used in the experiment. The average value of 3 trials is shown. It can be seen that the undifferentiation of hiPS cells is maintained by the addition of sulfated hyaluronic acid. It is a microscope picture of the colony of the human iPS cell 3 days after culture | cultivation. The left figure (A) shows the results when cultured without adding feeder cells and bFGF, and differentiation is observed. On the other hand, the right figure (B) shows the results when high sulfated hyaluronic acid is added, although no supporting cells and bFGF are added, and the undifferentiated state is maintained. Without adding feeder cells and bFGF, 10 μg / ml hyaluronic acid (HA), 10 μg / ml highly sulfated hyaluronic acid (molecular weight 100,000) (HA-HS) was added and cultured for 3 days, then Western blot analysis Were used to examine the expression level of each undifferentiated maintenance transcription factor (OCT4, KLF4, NANOG). The lower graph shows values after correcting the band intensity of each undifferentiated maintenance transcription factor with the band intensity of β-actin, which is an endogenous control. Two types of human iPS cell lines, hiPS # 16 (left) and hiPS # 25 (right), were used in the experiment. Each experiment was performed three times. The band shows only representative results. Significant differences were indicated by p ** <0.05 and p ** <0.01. This is a result of confirming undifferentiation by culturing human iPS cells using a culture substrate coated with sulfated hyaluronic acid and Matrigel. A colony of human iPS cells after 2 days in culture is shown. This is a result of adding sulfated hyaluronic acid to the culture medium and culturing for 3 days, and quantifying the number of cells and the amount of antibody (Ab. Conc) in the culture liquid by sandwich ELISA. As a control, no additive was added, and for comparison, hyaluronic acid and dermatan sulfate were added together.

Hereinafter, the present invention will be described with preferred methods and materials that can be used in the practice of the present invention, taking exemplary embodiments as examples. Unless otherwise noted in the text, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any materials and methods equivalent or similar to those described herein can be used as well in the practice of the invention. In addition, all publications and patents cited in this specification in relation to the invention described herein are hereby incorporated by reference, as examples of methods, materials, etc. that can be used with the invention. Part of it.

In this specification, when the expression “X to Y” is used, X is used as the lower limit and Y is used as the upper limit. In the present specification, “about” is used to mean ± 10%.

In the present invention, “sulfated hyaluronic acid” refers to a hydroxyl group of hyaluronic acid, which is a linear polymer polysaccharide formed by alternately binding β-DN-acetylglucosamine and β-D-glucuronic acid. This refers to substances in which part or all of them are substituted with sulfate groups.
The degree of sulfation is expressed as “degree of sulfation”. In the present specification, the degree of sulfation is represented by the sulfuric acid content (S content) (%) in the sulfated hyaluronic acid molecule, or the number of introduced sulfate groups (0 to 4) in the disaccharide unit which is a structural unit. Is done.
The sulfuric acid content (turbidimetric method) is determined by the Dogson-Price turbidimetric method (Dogson, KS and Price, RG, A note on the determination of the ester sulphate content of sulphated polysaccharides, Biochem J. vol 84 (1), 106- 110, 1962), and can be measured using potassium sulfate as a standard. The sulfuric acid content (weight method) can be measured using a method according to JIS JB. 2.17.3 Sulfate-weight method. The degree of sulfation can also be expressed as the average number of sulfate groups introduced. 0 to 4 sulfate groups can be introduced as disaccharide units. When expressed by the average number, the average number of sulfate groups in the disaccharide unit can be calculated from the S content based on the molecular weight of sulfated hyaluronic acid.
The sulfated hyaluronic acid used in the present invention (hereinafter sometimes simply referred to as “sulfated hyaluronic acid”) is hyaluronic acid having a relatively high degree of sulfation. As a specific compound of sulfated hyaluronic acid, hyaluronic acid having a sulfated degree of 1.0 or less as a sulfate group / 2 monosaccharide has been reported (see, for example, JP-A-2009-278873). ). The sulfated hyaluronic acid used in the present invention has an S content of about 7% or more by the turbidimetric method, and the number of sulfate groups / 2 monosaccharide is about 1.3 or more.

According to the culture method of the present invention, sulfated hyaluronic acid used when culturing pluripotent stem cells without using supporting cells has an average number of introduced sulfate groups of about 1.3 or more, preferably about 1.5 or more, more preferably about 2.0 or more, and further preferably about 2.5 or more.
According to the culture method of the present invention, the sulfated hyaluronic acid used in the case of culturing pluripotent stem cells without using supporting cells and bFGF has an average number of introduced sulfate groups of about 1.3 or more, preferably Is about 1.5 or more, more preferably about 2.0 or more, and still more preferably about 3.0 or more.
According to the culture method of the present invention, pluripotent stem cells can be cultured without using supporting cells and bFGF, and the pluripotent stem cells are maintained in an undifferentiated state or in a state where pluripotency is maintained. Can be cultured. However, culture in a medium containing sulfated hyaluronic acid and containing bFGF is also included in the present invention and is part of the present invention.

Sulfated hyaluron used in the cell culture substrate of the present invention coated with sulfated hyaluronic acid, used for culturing pluripotent stem cells that do not use feeder cells, and in addition to feeder cells, and in addition, do not use bFGF. The acid is sulfated hyaluronic acid having an average number of introduced sulfate groups of about 1.3 or more, preferably about 1.3 to about 3.0.
In the above culture, the cell culture substrate of the present invention used is preferably a cell culture substrate coated with the sulfated hyaluronic acid together with an extracellular matrix.
Using the cell culture substrate of the present invention coated with sulfated hyaluronic acid, pluripotent stem cells can be cultured without using supporting cells and bFGF, Can be cultured in a state of maintaining pluripotency or maintaining pluripotency. However, the use of the cell culture substrate of the present invention in a medium containing bFGF is not excluded, and the cell culture substrate of the present invention used for culture in a medium added with bFGF is also included in the present invention. And is part of the present invention.

The sulfated hyaluronic acid used when culturing a hybridoma capable of producing an antibody according to the culture method of the present invention has a sulfation degree of about 7% or more, preferably about 10% or more in terms of sulfuric acid content. Preferably it is about 13% or more, more preferably about 14% or more.
The sulfated hyaluronic acid used when culturing a hybridoma capable of producing an antibody according to the culture method of the present invention has an average number of introduced sulfate groups of about 1.3 or more, preferably about 1.5 or more. More preferably, it is about 2.5 or more, more preferably about 3.0 or more, and still more preferably about 3.5 or more.

Sulfated hyaluronic acid can be obtained by dissolving hyaluronic acid and a sulfating agent in an appropriate solvent using a known sulfation reaction and reacting under heating. The degree of sulfation can be adjusted by appropriately changing the amount of sulfating agent used with reference to known methods. The degree of sulfation can be measured by, for example, the method described above, but can also be determined by quantifying sulfate ions generated by acid hydrolysis by ion chromatography.

As a solvent used in the sulfation reaction, a solvent that can be generally used in the technical field can be used without limitation. For example, N, N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, N, N-dimethylacetamide, Examples thereof include pyridine, N, N-dimethylacrylamide, or a mixed solvent thereof.

As the sulfating agent, a sulfating agent generally used in the art can be used without limitation. For example, anhydrous sulfuric acid and pyridine, picoline, 2,6-lutidine, trimethylamine, triethylamine, N, N-dimethylformamide, dioxane And the like, or sulfuric acid-dicyclohexylcarbodiimide, chlorosulfone, and the like. In the sulfation reaction, an acid catalyst such as trifluoroacetic acid or trifluoromethanesulfonic acid may be added to the reaction system.

The reaction temperature and reaction time of the sulfation are not particularly limited, and examples thereof include 0 to 100 ° C. and 5 minutes to 5 days.

The molecular weight of sulfated hyaluronic acid that can be used in the present invention is not particularly limited, and any molecular weight of sulfated hyaluronic acid can be used. In the present invention, as shown in the following examples, low molecular weight hyaluronic acid (for example, having an average molecular weight of about 100,000), high molecular weight hyaluronic acid (for example, having an average molecular weight of 600,000) Either can be used. In the present invention, when referring to the molecular weight of sulfated hyaluronic acid, it means the molecular weight indicated by any of the display methods generally used in the technical field, and is not limited to this, for example, kinematic viscosity of hyaluronic acid as a raw material The molecular weight calculated from can be used.

The substrate for cell culture of the present invention can be produced by coating sulfated hyaluronic acid on the surface of the substrate for cell culture. As a substrate for cell culture, a substrate for cell culture used in the technical field relating to cell culture can be used without limitation. For example, a substrate used for a petri dish or plate for animal cell culture. Materials can be used.
The material that can be such a base material is not limited to this, and examples thereof include polypropylene, polystyrene, high density polyethylene, polyethylene terephthalate copolymer, polycarbonate, polyethersulfine, and MPC polymer. .

In the present invention, “the surface is coated with sulfated hyaluronic acid” means a state in which the sulfated hyaluronic acid is physically bound or held on the surface of the cell culture substrate. Including the state. The term “physically bound” refers to a state in which they are bound non-covalently or covalently, and is not limited thereto, but, for example, it is bound in an ionic manner, directly or via a linker or the like. You can raise the state. The means for covalent bonding can be appropriately selected according to the physicochemical properties of the substrate surface and the reactive groups presented on the surface. For example, it can be linked directly or via a linker using a photoreactive crosslinking agent. The state in which sulfated hyaluronic acid is retained on the substrate surface is not limited to this. For example, based on the affinity between sulfated hyaluronic acid and the substrate, sulfated hyaluronic acid is retained on the substrate surface. You can raise the state. For example, by applying a solution or solvent containing sulfated hyaluronic acid to the surface of the substrate, and then removing the solution or solvent, a cell culture substrate coated with sulfated hyaluronic acid on the surface is prepared. Can do. The method for removing the solution or the solvent is not particularly limited, and for example, room temperature drying, high temperature drying, reduced pressure drying and the like can be used.
In addition, when the surface is coated with a polymeric substance such as an extracellular matrix, the sulfated hyaluronic acid is retained on the substrate surface while the sulfated hyaluronic acid is retained in the polymeric substance such as the extracellular matrix. The state that has been done is also included.

The amount of sulfated hyaluronic acid coated on the surface of the cell culture substrate of the present invention depends on the purpose of the culture, as compared to the case where the cells are cultured on a substrate not coated with sulfated hyaluronic acid. For example, the amount is about 0.05 μg / cm ² or more, preferably about 0.5 μg / cm ² or more, more preferably about 1 μg / cm ² or more, and still more preferably. Can be about 2 μg / cm ² or more. The thickness of the sulfated hyaluronic acid coated on the surface is not particularly limited, but can be appropriately selected in consideration of the purpose of use, preparation method, cost and the like.

The base material for cell culture of the present invention also includes a material obtained by coating the surface thereof with a sulfate, hyaluronic acid and a high-molecular substance such as a protein, a polymer or an extracellular matrix. These macromolecules serve to retain sulfated hyaluronic acid, but are not limited thereto. As the protein that can be used, any protein that does not inhibit cell culture / proliferation can be used without limitation. For example, albumin, transferrin, collagen, fibronectin, Fc fusion protein and other chimeric proteins, and cell adhesion support Peptide fragments and the like. The polymer that can be used is not particularly limited as long as it does not inhibit cell culture and proliferation, and examples thereof include polylysine, polylactic acid, polyglycolic acid, and a copolymer of lactic acid and glycolic acid. Can do. As the polymer substance coated with sulfated hyaluronic acid, an extracellular matrix is preferable. By coating the extracellular matrix together with hyaluronic acid sulfate, the efficiency of cell culture and proliferation can be further increased. Examples of the extracellular matrix include matrigel, laminin, laminin 511, proteoglycan and the like. The amount of the extracellular matrix coated on the surface of the cell culture substrate of the present invention can be appropriately selected according to the cells used, the purpose of use, the culture conditions, and the like. For example, when using Matrigel, cells using Matrigel are used. You can refer to reports on culture.

In the cell culture substrate of the present invention, the method for coating sulfated hyaluronic acid with a polymer substance is not particularly limited. For example, in the case of coating with an extracellular matrix, but not limited thereto, a mixed solution is prepared by adding sulfated hyaluronic acid to a medium in which the extracellular matrix (eg, Matrigel) is dissolved, and then the mixed solution is added to the culture medium. It can be prepared by applying to the surface of the material and adding it to a culture dish, for example. The cell culture substrate coated with sulfated hyaluronic acid and extracellular matrix prepared as described above (for example, a culture petri dish) may be used as it is, but may be used after storage until use. it can. The storage method can be appropriately selected according to the extracellular matrix used for the coating, and examples include normal temperature, refrigeration, and frozen storage. For example, when Matrigel is used as the extracellular matrix, refrigeration or frozen storage is desirable. In use, the medium containing the extracellular matrix that is not coated on the surface and the sulfated hyaluronic acid is removed, and then the medium and cells are added for culturing.

In the cell culture substrate of the present invention, when sulfated hyaluronic acid is coated on the surface together with a polymer substance (for example, protein, polymer or extracellular matrix), sulfated hyaluronic acid is uniformly distributed in the polymer substance. It is preferable to hold in a dispersed state. If the concentration of the sulfated hyaluronic acid in the mixture is too high, the sulfated hyaluronic acid is not preferable because it is agglomerated with each other or stretched into a fiber. The amount of sulfated hyaluronic acid retained in the polymer substance can be adjusted, for example, by appropriately selecting the concentration of sulfated hyaluronic acid in the mixed solution before being added to a culture substrate such as a petri dish. Thus, the cell culture substrate of the present invention coated with sulfated hyaluronic acid uniformly dispersed in a polymer substance can be prepared.

The cell culture substrate according to one embodiment of the present invention includes a cell culture substrate coated with sulfated hyaluronic acid on the surface, and a sulfated hyaluronic acid polymer material such as an extracellular matrix such as matrigel, protein Alternatively, any cell culture substrate coated with a polymer is included. A cell culture substrate having a surface coated with sulfated hyaluronic acid can be produced in a sterile state or sterilized after production. The sterilization treatment can be appropriately selected by those skilled in the art according to the components used together with the sulfated hyaluronic acid. For example, the sterilization treatment method is not limited to this, but for example, electron beam, gamma ray, ethylene oxide, High pressure steam (autoclave) can be raised. The cell culture substrate coated with sulfated hyaluronic acid together with an extracellular matrix such as matrigel is preferably produced in a sterile state according to a conventional method.

The cell culture substrate of the present invention can also be used in a part of a container, instrument or apparatus for cell culture. Such culture vessels, instruments and devices are also included in the present invention. As the container, instrument, and apparatus for cell culture, those generally used for cell culture can be used without limitation.

The present invention is also a method for culturing a cell by culturing and proliferating a pluripotent stem cell while maintaining an undifferentiated state or having a differentiation potential using the cell culture substrate of the present invention.

Examples of the pluripotent stem cells used in the culture using the cell culture method of the present invention or the cell culture substrate of the present invention include ES cells and iPS cells. Preferably, human ES cells or human iPS cells are used. A cell, particularly preferably a human iPS cell.
Cell culturing conditions can be appropriately selected according to the type of cells intended for culturing and the purpose of culturing, and such culturing conditions have been reported in various ways.
When the cell culture method or cell culture substrate of the present invention is used, pluripotent stem cells can be cultured without using supporting cells (feeder cells). In addition, when pluripotent stem cells are cultured using the cell culture substrate of the present invention, the amount of bFGF that is normally required to maintain an undifferentiated state can be reduced or eliminated. .
That is, the present invention, as one aspect thereof, in a case where pluripotent stem cells are not differentiated or have a differentiation ability, without using a supporting cell layer for supporting cell proliferation, in some cases as supporting cells. In addition, a culture method for stably and consistently growing without using bFGF is provided.

The present invention also includes a culture method for the production of an antibody using a sulfated hyaluronic acid having a relatively high degree of sulfation. According to the culture method of the present invention, antibody production ability can be remarkably increased by culturing a hybridoma having antibody production ability together with sulfated hyaluronic acid.
The sulfated hyaluronic acid that can be used in the culture for antibody production according to the present invention is a sulfated hyaluronic acid having a relatively high degree of sulfation as described above. Specifically, the S content is 7% or more (preferably 10% or more, more preferably 13% or more, more preferably 14% or more) sulfated hyaluronic acid having a degree of sulfation, or an average of at least 1 sulfate group with respect to a disaccharide unit as a constituent unit. 3 or higher (preferably 2.0 or higher, more preferably 2.5 or higher, still more preferably 3.0 or higher, and still more preferably 3.5 or higher). Both low molecular weight sulfated hyaluronic acid and high molecular weight sulfated hyaluronic acid can be used. In the culture method for producing the antibody of the present invention, the amount of sulfated hyaluronic acid added to the medium can be appropriately selected depending on the cells used, culture conditions, etc., but is not limited thereto, for example, It is added to the medium at a concentration of 0.1 to 100 μg / mL, preferably 1 to 100 μg / mL. The medium is not particularly limited, and any medium that can be used in antibody production using a hybridoma can be used. For example, RPMI-1640 medium, Dulbecco's modified Eagle medium, Nutrient Mixture F-12 Ham, Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham, Iscove's Modified Dulbecco's Medium, GIT medium. In addition, the culture conditions of the hybridoma producing the antibody can be appropriately determined by referring to a method known per se.
The present invention also includes a method of culturing cells having antibody production ability using the cell culture substrate of the present invention. When cells capable of producing an antibody are cultured using the cell culture substrate of the present invention, the amount of antibody produced can be remarkably increased.
Therefore, the present invention provides, as another aspect, a cell culture method and a cell culture substrate for producing an antibody by culturing antibody-producing cells.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of Sulfated Hyaluronic Acid Sulfated hyaluronic acid was prepared by sulfating hyaluronic acid. For sulfation of hyaluronic acid, known sulfation reactions were used, and various sulfation degrees were produced by appropriately adjusting the conditions. Hyaluronic acid used for the sulfation was one having a molecular weight of 100,000 (hereinafter referred to as low molecular weight hyaluronic acid) and 600,000 (hereinafter referred to as high molecular weight hyaluronic acid).
Preparation of sulfated hyaluronic acid was first done at a small level (amount of hyaluronic acid used, 1 g) and then at a scaled up level (amount of hyaluronic acid used, 20 g).
The degree of sulfation was confirmed for the prepared sulfated hyaluronic acid. The results are shown below. Table 1 shows the results of measuring the degree of sulfation of sulfated hyaluronic acid prepared using low molecular weight hyaluronic acid, and Table 2 shows the degree of sulfidation of sulfated hyaluronic acid prepared using high molecular weight hyaluronic acid. It is a result. “S content (turbidimetric method)” is a result measured by the Dogson-Price turbidimetric method, and “S content (gravimetric method)” is a method according to JIS JB. 2.17.3 Sulfate-weight method. It is the result of having measured using. The “sulfation degree” is the number of sulfate groups introduced by the average number per disaccharide unit.

The sulfated hyaluronic acid used in the present invention has a relatively high degree of sulfation. In the present specification, low sulfated hyaluronic acid having a relatively high degree of sulfation, of which the degree of sulfation is low, specifically, the degree of sulfation (number of sulfate groups / 2 monosaccharides) is about 1. Low sulfated hyaluronic acid having a degree of sulfation of about 5, high sulfated hyaluronic acid having a high degree of sulfation, specifically, high sulfated hyaluronic acid having a degree of sulfation (number of sulfate groups / 2 monosaccharides) of 3 or more Called acid. The highly sulfated hyaluronic acid shown in the above table indicates that most of the hydroxyl groups that can be substituted with sulfate groups are sulfated.

Example 2: Culture of iPS / ES cells In order to culture and expand human iPS cells while maintaining their undifferentiated state, culture on mouse-derived feeder cells and addition of bFGF to the medium is necessary. When cultured in the absence of both, human iPS cells differentiate rapidly. Therefore, the necessity of addition of both of these is an obstacle to culturing a large amount of iPS cells that have maintained undifferentiation.
Therefore, it was examined whether or not human iPS cells can be cultured and proliferated while maintaining undifferentiated state in the absence of supporting cells and bFGF by adding the highly sulfated hyaluronic acid prepared in Example 1 to the medium.
As human iPS cells (hiPSC), MRC-hiPS_Fetch (cell number: NIHS0604) and MRC-hiPS_Tic (cell number: JCRB1331) (both described in Exp. Cell Res. 315 (2009) 2727-2740, from the National Center for Growth Medicine) Used). On each MEFs inactivated with 10 μg / mL mitomycin C (manufactured by Sigma) in iPSellon (manufactured by Cell-Sight) with bFGF (manufactured by Wako Pure Chemical Industries) at a concentration of 10 ng / mL. Maintained at.
The above-mentioned hiPS cells (hiPS # 16 and hiPS # 25) were cultured in hiPS cells (with a molecular weight of 100,000) added with high sulfated hyaluronic acid (molecular weight 100,000) to a concentration of 10 μg / ml in the absence of feeder cells and bFGF. DMEM / F-12 (Gibco), 20% KnockOut Serum Replacement XenoFree (Thermo Fisher Scientific), 0.1 mM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 1% Penicillin-Streptomycin (Thermo Fisher Scientific), 0.1 mM 2- In addition to Mercaptoethanol (Gibco) and 2 mM L-Glutamine (Gibco)), this was used to culture on a culture dish treated with matrigel (BD Bioscience) for 3 days. Three days later, the number of colonies of undifferentiated hiPS cells formed was counted and analyzed quantitatively. The results are shown in FIG. Compared to other conditions, the number of undifferentiated hiPS cell colonies was significantly increased in the medium supplemented with highly sulfated hyaluronic acid. The photograph of the formed colony in each condition of the presence or absence of sulfated hyaluronic acid is shown in FIG. A shows the results when the cells were cultured without adding the feeder cells and bFGF, and B shows the results when the cells were not added with the feeder cells and bFGF but highly sulfated hyaluronic acid was added. By adding highly sulfated hyaluronic acid, it was confirmed that the undifferentiation of hiPS cells was maintained even in the absence of feeder cells and bFGF.

Example 3: Confirmation of the expression of undifferentiated factors For hiPS cells cultured in a medium supplemented with sulfated hyaluronic acid, the expression levels of transcription factors (OCT4, KLF4, NANOG) essential for maintaining undifferentiated properties were measured by immunoblotting. As a result, undifferentiation was confirmed. The results are shown in FIG.
It was suggested that highly sulfated hyaluronic acid in the medium promotes the expression level of the undifferentiated maintenance transcription factor even in the absence of supporting cells and bFGF, and contributes to the maintenance of undifferentiated state.

Example 4 Adhesion of Sulfated Hyaluronic Acid to Culture Substrate Sulfated hyaluronic acid was adhered to a cell culture substrate, and the amount of adhesion was evaluated.
Sulfated hyaluronic acid was adhered to various cell culture substrates with different surface treatments, and the amount of adhesion was confirmed. As cell culture substrate, BD (Becton Dickinson) cell culture treatment petri dish (35mm, polystyrene, hydrophilic treatment by vacuum gas plasma treatment), BD untreated petri dish (35mm, polystyrene, hydrophobic surface), BD NH ₂ treatment (6 well, polystyrene, amino group addition surface), BD COOH (6 well, polystyrene, carboxyl group addition surface), cell culture treatment (6 well, polystyrene, hydrophilic treatment) manufactured by IWAKI, Sumitomo Bakelite Cell culture treatment (6 well, polystyrene, hydrophilic treatment), untreated (6 well, polystyrene, hydrophobic surface) manufactured by Sumitomo Bakelite Co., Ltd. was used. As the sulfated hyaluronic acid, a low molecular weight high sulfated hyaluronic acid was used. Specifically, sulfated hyaluronic acid was dissolved in PBS buffer and kept at 37 ° C. for 1 hour, and sulfated hyaluronic acid that did not adhere was washed with PBS. Adhesion amount of sulfated hyaluronic acid was evaluated by DMMB method (Connect Tissue Res. 1982; 9 (4): 247-8. A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Farndale RW, Sayers CA, Barrett AJ.) did. The results are shown in the table below. The adhesion amount was the sulfated sugar chain adhesion amount (μg) per area (9.6 cm ² ). 100 μg / mL and 10 μg / mL are treatment concentrations at the time of coating. u. d. Is below the measurement limit

Adhesion to a culture substrate to which an amino group was added was good.

Next, adhesion of the sulfated sugar chain to the culture substrate by covalent bonding was examined. Specifically, sulfated sugar chains were covalently bound to a culture substrate using a photoreactive diazirine compound as follows.
SDA (NHS-Diazirine) was dissolved at a concentration of 50 mM, added to a culture petri dish having an amino group, reacted at room temperature for 30 minutes, and diazirine was bound to the petri dish surface. To this, a sulfated sugar chain (chondroitin sulfate D) was added and bound to the substrate surface by UV irradiation. As a result, adhesion of the sulfated sugar chain to the culture substrate (petri dish) by covalent bond could be confirmed. Thus, it was found that a sulfated sugar chain containing sulfated hyaluronic acid can be bound to a BD NH ₂ 6 well plate or the like by a method using a photoreactive dilysine compound.

Example 5: Coating of Sulfated Hyaluronic Acid and Matrigel on Culture Substrate A culture substrate coated with a cell matrix matrix in addition to sulfated hyaluronic acid was prepared, and culture using it was examined.
A mixed coating of a basement membrane matrix (Matrigel (registered trademark), Corning) used for iPS cell culture and sulfated hyaluronic acid was performed. As the sulfated hyaluronic acid, a low molecular weight (molecular weight 100,000) sulfated hyaluronic acid was used. Specifically, the culture substrate was coated as follows. Dissolve Matrigel to 2.5% in cell culture medium DMEM, add sulfated hyaluronic acid to 2 μg / ml, 10 μg / ml or 100 μg / ml and mix, then mix Was incubated at 37 ° C. and 5% CO ₂ for 1 hour. The petri dish was frozen and stored until use, and the cell culture medium containing Matrigel was extracted before use.
In order to confirm the sulfated hyaluronic acid concentration on the surface of the prepared culture substrate, sulfated sugar was quantified by DMMB method. As a result, the sulfated hyaluronic acid concentration per 9 cm ^{2 of the} culture substrate surface area was 0.96 μg, 5.11 μg, and 8.08 μg, respectively, when the coating amount was 2 μg / ml, 10 μg / ml, or 100 μ / ml. It was. Moreover, as a result of confirming the surface state with a microscope, it was confirmed that the sulfated hyaluronic acid was uniformly dispersed in the matrigel when the coating addition amount was 10 μg / ml.

Using a culture dish coated with Matrigel mixed with 10 μg / mL sulfated hyaluronic acid, the cells were cultured without adding bFGF. Specifically, only Matrigel without adding feeder cells and bFGF, 10 μg / mlＨhyaluronic acid (HA) mixed matrigel, 10 μg / ml low sulfated hyaluronic acid (HA-LS) mixed matrigel, 10 μg / ml high sulfated HiPS cells (hiPS # 16 and hiPS # 25) were respectively seeded and cultured on a plate coated with hyaluronic acid (HA-HS) mixed matrigel. As a result, a colony maintaining undifferentiated properties was observed on the second day of culture regardless of whether low sulfated hyaluronic acid or high sulfated hyaluronic acid was used. The results are shown in FIG.

Example 6: Enhancement of antibody production ability The effect of enhancement of antibody production ability by sulfated hyaluronic acid was confirmed.
Sulfated hyaluronic acid was prepared in Example 1, low molecular weight low sulfated hyaluronic acid (molecular weight 100,000) (HA-LS), low molecular weight high sulfated hyaluronic acid (molecular weight 100,000) (HA-HS). Was used. As comparative examples, unsulfated hyaluronic acid (HA) and dermatan sulfate were used.
As the medium, UltraDoma-PF medium (purchased from LONZA) was used, and hyaluronic acid, low and high sulfated hyaluronic acid, and dermatan sulfate were added to a concentration of 10 μg / ml.
The cell used was GGR12 strain which is a hybridoma producing IgG.
Antibody production was performed according to conventional methods. Specifically, the hybridoma was seeded at a concentration of ⁴ × 10 ⁴ cells / ml, cultured for 3 days in a medium supplemented with sulfated hyaluronic acid, and the number of cells and the amount of antibody in the culture solution were measured. The amount of antibody (antibody concentration) was measured by sandwich ELISA using an anti-IgG polyclonal antibody. The results are shown in FIG.
There was no difference in the number of cells (cell proliferation) even when cultured in a medium to which any compound was added. However, the amount of antibody production was significantly increased in the medium supplemented with sulfated hyaluronic acid, and increased three times or more in the medium supplemented with highly sulfated hyaluronic acid.

The above detailed description merely illustrates the object and object of the present invention and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.

When the culture method of the present invention is used, hPSCs are cultured in an undifferentiated state or in a state in which pluripotency is maintained without using support cells (feeder cells) that hinder the production of large-scale cells. It becomes possible to proliferate. In addition, the culture method of the present invention can increase the amount of antibody produced by a hybridoma.

Claims

Cell culture substrate with sulfated hyaluronic acid coated on the surface.
2. The cell culture substrate according to claim 1, wherein the sulfated hyaluronic acid is sulfated hyaluronic acid having a sulfation degree of 7% or more in S content.
2. The cell culture substrate according to claim 1, wherein the sulfated hyaluronic acid is introduced at a ratio of at least 1.3 or more on average with respect to a disaccharide unit as a structural unit.
The cell culture substrate according to claim 3, wherein the sulfated hyaluronic acid is introduced at a ratio of 1.3 to 3.0 on average with respect to a disaccharide unit as a structural unit.
The base material for cell culture according to claim 3, wherein the sulfated hyaluronic acid is introduced with an average of 3.0 or more sulfate groups with respect to the disaccharide unit as a structural unit.
A cell culture medium according to any one of claims 1 to 5, which is a substrate for culturing pluripotent stem cells and is used for culturing to maintain pluripotent stem cells in an undifferentiated state. Base material.
The cell culture substrate according to claim 6, wherein the pluripotent stem cells are iPS cells.
The cell culture substrate according to any one of claims 1 to 7, wherein the sulfated hyaluronic acid is coated on the surface together with an extracellular matrix, protein or polymer.
The cell culture substrate according to claim 8, wherein the sulfated hyaluronic acid is present in a state of being uniformly dispersed in an extracellular matrix, a protein or a polymer.
A cell culture instrument comprising the cell culture substrate according to any one of claims 1 to 9.
A method for culturing pluripotent stem cells, comprising culturing pluripotent stem cells using the cell culture substrate according to any one of claims 1 to 9 and maintaining their differentiation ability.
A method for culturing pluripotent stem cells comprising the following steps:
A step of culturing cells using a culture medium to which sulfated hyaluronic acid is added under culture conditions not containing feeder cells, wherein the sulfated hyaluronic acid has a sulfation degree of 7% or more in S content. ,
A culture method comprising:
A method for culturing pluripotent stem cells comprising the following steps:
A step of culturing cells under a culture condition without supporting cells using a medium to which sulfated hyaluronic acid is added, wherein the sulfated hyaluronic acid has a sulfate group with respect to a disaccharide unit as a constituent unit; Introduced at an average rate of at least 1.3,
A culture method comprising:
The culture method according to claim 12 or 13, wherein the culture condition is a culture condition not further containing bFGF.
The culture method according to any one of claims 12 to 14, wherein the culturing step is performed in the presence of an extracellular matrix, a protein, or a polymer.
The culture method according to any one of claims 12 to 15, wherein the pluripotent stem cells are human iPS cells.
A method for producing an antibody by culturing a hybridoma having the ability to produce an antibody, comprising the following steps:
Culturing cells in a medium supplemented with sulfated hyaluronic acid, wherein the sulfated hyaluronic acid has a degree of sulfation of 7% or more in S content,
A method comprising the steps of:
The method according to claim 17, wherein the degree of sulfation is 13% or more.
A method for producing an antibody by culturing a hybridoma having the ability to produce an antibody, comprising the following steps:
A step of culturing cells in a medium to which sulfated hyaluronic acid is added, wherein the sulfated hyaluronic acid is introduced at a ratio of at least 1.3 or more on average with respect to the disaccharide unit as a constituent unit. The degree of sulfation being
A method comprising the steps of:
The method according to claim 19, wherein the degree of sulfation is introduced at a ratio of 3.0 or more on the average of sulfate groups.