JP2609549B2 - Cell culture substrate - Google Patents
Cell culture substrateInfo
- Publication number
- JP2609549B2 JP2609549B2 JP1252061A JP25206189A JP2609549B2 JP 2609549 B2 JP2609549 B2 JP 2609549B2 JP 1252061 A JP1252061 A JP 1252061A JP 25206189 A JP25206189 A JP 25206189A JP 2609549 B2 JP2609549 B2 JP 2609549B2
- Authority
- JP
- Japan
- Prior art keywords
- polysaccharide
- cell culture
- substrate
- culture substrate
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、細胞を培養する際に用いる培養基質に関す
るものである。Description: TECHNICAL FIELD The present invention relates to a culture substrate used for culturing cells.
一般的に言つて、細胞は、浮遊状態で増殖・分化する
ものと、基質に接着した状態でなければ増殖・分化を行
なわないものとに大別できる。接着製細胞にとつてはそ
の基質は、生存に必須というだけでなく、機能発現にも
大きな意味あいを持つている。Generally speaking, cells can be broadly classified into those that grow and differentiate in suspension and those that do not grow or differentiate unless they are attached to a substrate. For adherent cells, the substrate is not only essential for survival, but also has significant implications for functional expression.
培養細胞の環境を構成する一因としての基質につい
て、従来は、単に、細胞が載つている材料、程度の認識
しかなかつた。しかしながら、細胞生物学の研究の進展
に伴い、細胞本来の機能を発現させるためには基質の役
割が重要である事が多くの研究者に認められてきた。Heretofore, as to a substrate that constitutes an environment of a cultured cell, conventionally, only the material on which the cell is mounted and the degree of its recognition have been recognized. However, with the progress of research in cell biology, many researchers have recognized that the role of the substrate is important for expressing the original functions of cells.
細胞を培養する基質として最初はガラスが使われた。
しかし、ガラス製基質は再使用するための洗浄のわずら
わしさや重くて扱いにくいという欠点があつた。現在で
は、表面処理を施されたプラスチツク製培養基質が開発
され、従来のガラス製基質はほとんど使われなくなつ
た。このプラスチツク製培養基質は、普通の細胞なら
ば、だいたいは培養可能であること、軽くて使い易いこ
と、安価であることなどから幅広く用いられている。Glass was initially used as a substrate for culturing cells.
However, glass substrates have the disadvantage that they are cumbersome to wash for reuse and are heavy and unwieldy. At present, surface-treated plastic culture substrates have been developed, and conventional glass substrates have become almost obsolete. This plastic culture substrate is widely used for ordinary cells because it can be generally cultured, is light and easy to use, and is inexpensive.
現在、住友ベークライト社,Corning社,Costar社,Bect
on Dickinson社などから各種の形態の製品群が上市され
ている。Currently, Sumitomo Bakelite, Corning, Costar, Bect
Various forms of products are on the market from on Dickinson and others.
しかし、これらガラス，プラスチツク製基質では、必
ずしもすべての細胞の機能を良好に維持したまま培養で
きるわけではなく、増殖しないこと、機能が発現しない
こと、分化が不充分であることなどの現象がしばしば認
められる。However, with these glass and plastic substrates, it is not always possible to culture cells while maintaining the functions of all cells in a good condition, and phenomena such as non-proliferation, non-expression of functions, and insufficient differentiation are often observed. Is recognized.
たとえば、神経系細胞，乳腺細胞、肝細胞，腎細胞な
どでは、その傾向が顕著にあらわれる。細胞は生体の中
では細胞外マトリツクスという複雑な構造体に囲まれて
生存している。For example, in nervous system cells, mammary gland cells, hepatocytes, kidney cells and the like, the tendency is remarkable. Cells survive in living organisms surrounded by a complex structure called extracellular matrix.
この構造体はコラーゲン，フイブロネクチン，ヒアル
ロン酸などの多種類の成分からなり、細胞の分化・増殖
を制御している事が明らかにされつつある。This structure is composed of various components such as collagen, fibronectin, and hyaluronic acid, and it has been revealed that the structure controls the differentiation and proliferation of cells.
上記したガラス，プラスチツク製基質では培養が困難
な細胞を培養する際にはより生体環境に近づけた天然物
を用いる手法が必要とされる。具体的には、コラーゲ
ン，ポリアミド酸などをガラス，プラスチツク基質上に
被覆して培養する必要がある。When culturing cells that are difficult to culture with the above-mentioned glass and plastic substrates, a technique using a natural product that is closer to the biological environment is required. Specifically, it is necessary to coat collagen, polyamic acid, or the like on a glass or plastic substrate and culture.
このような形態で培養するとある程度の機能発現が見
られる事が知られている。It is known that when the cells are cultured in such a form, a certain degree of function expression is observed.
しかしながら、この手法では被覆した被覆層がはがれ
たり、溶出したりして安定した培養ができないこと、高
価であること、使い易いものではないこと、などの問題
点があつた。However, in this method, there were problems such as that the coated coating layer was peeled off or eluted, so that stable culture could not be performed, it was expensive, and it was not easy to use.
そこで、本発明者は、細胞の機能を良好に維持する安
定な培養基質を得るため、種々の天然材料と表面加工技
術を総合的に検討した結果、細胞の機能を良好に維持し
得る優れた基質を見い出し、本発明を完成するに至つた
ものであり、その目的とする所は、従来のコラーゲンや
ポリアミド酸被覆基質とは全く異るスルホン基を有する
多糖を主成分とする新規培養基質を提供するにある。Therefore, the present inventor comprehensively studied various natural materials and surface processing techniques in order to obtain a stable culture substrate that maintains the function of the cell satisfactorily. The present invention has led to the completion of the present invention.The purpose of the present invention is to provide a novel culture substrate mainly composed of a polysaccharide having a sulfone group completely different from a conventional collagen or polyamic acid-coated substrate. To offer.
本発明は、スルホン基を有する多糖、スルホン基を有
する多糖とゼラチンからなる組成物、スルホンを有する
多糖とアミノ基誘導体を有する多糖からなる組成物を、
それぞれ低温プラズマ処理して得られる細胞培養用基質
に係るものである。The present invention provides a polysaccharide having a sulfone group, a composition comprising a polysaccharide having a sulfone group and gelatin, a composition comprising a polysaccharide having a sulfone and a polysaccharide having an amino group derivative,
The present invention relates to a cell culture substrate obtained by low-temperature plasma treatment.
本発明に用いられるスルホン基を有する多糖とは、ヘ
パリン，ヘパラン硫酸，コンドロイチン硫酸，ケラタン
硫酸，カラゲナンなどである。その由来は動物，植物，
合成品のいずれでもよく特に制限されない。ゼラチンも
由来は問わないが、生化学用として使われているものが
好ましい。食品用のものは不純物が多く好ましくない。
アミノ基誘導体を有する多糖としては、たとえば、キチ
ン，キトサン塩酸塩，ヒアルロン酸，コンドロイチン，
テイクロン酸などがある。天然物，合成品，いずれを用
いてもよい。これらの組成物を作る方法は、粉末状にし
て混和する。水溶液にして混和するなど、各成分が変質
しない限り、いかなる手法でもよいが、水溶液にする方
法が好ましい。The polysaccharide having a sulfone group used in the present invention includes heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, carrageenan and the like. The origin is animals, plants,
Any of synthetic products may be used without any particular limitation. Gelatin may be of any origin, but is preferably used for biochemistry. Food products are undesirable because they contain many impurities.
Examples of the polysaccharide having an amino group derivative include chitin, chitosan hydrochloride, hyaluronic acid, chondroitin,
Teichulonic acid and the like. Any of natural products and synthetic products may be used. The method of making these compositions is to powder and mix. Any method may be used as long as each component does not deteriorate, such as mixing with an aqueous solution, but a method of preparing an aqueous solution is preferable.
組成物の比率は、それぞれ５〜95％までの間であれば
よい。培養基質とするためには、デイツシユ，フイル
ム，薄膜，マイクロキヤリヤ状、などの形状にする必要
があるが、いずれの場合も好ましくは水溶液状にしたも
のを用いるのがよい。成型する、キヤストする、有機溶
媒中へ適下するなどの手法を用いる事により容易に培養
基質となす事ができる。The proportions of the compositions may each be between 5 and 95%. In order to use it as a culture substrate, it is necessary to make it into a shape such as a dish, a film, a thin film, or a microcarrier. In any case, it is preferable to use an aqueous solution. It can be easily used as a culture substrate by using techniques such as molding, casting, or dropping into an organic solvent.
このようにして調製したものを低温プラズマで処理す
る。低温プラズマ処理は、試料を入れた反応容器内を減
圧後ガスを導入し、0.01〜0.1Torrに保ち、ここで電圧
を印加し、５〜100Wの電力で３〜60分反応させる。用い
るガスは、アルゴン、ネオンなどの不活性ガス、一酸化
炭素，二酸化炭素，酸素，水素，窒素，アンモニア，水
蒸気又はこれらの混合物を用いる事ができるが、好まし
くは一酸化炭素，アンモニアである。この低温プラズマ
処理を施す事により表層構造変化のすなわち、架橋，再
重合，官能基の導入などが起こり、安定で培養機能に優
れた培養基質が形成される。The material thus prepared is treated with low-temperature plasma. In the low-temperature plasma treatment, a gas is introduced into the reaction vessel containing the sample after the pressure is reduced, and the gas is maintained at 0.01 to 0.1 Torr. A voltage is applied here, and the reaction is performed for 3 to 60 minutes with a power of 5 to 100 W. As a gas to be used, an inert gas such as argon or neon, carbon monoxide, carbon dioxide, oxygen, hydrogen, nitrogen, ammonia, water vapor or a mixture thereof can be used, but carbon monoxide and ammonia are preferable. By performing this low-temperature plasma treatment, a change in the surface layer structure, that is, crosslinking, repolymerization, introduction of a functional group, and the like occur, and a culture substrate that is stable and excellent in culture function is formed.
本発明に係る細胞培養用基質は、従来の基質と異り細
胞の分化機能の発現に高い効果を示す特徴を有する。The substrate for cell culture according to the present invention is different from the conventional substrate in that it has a feature that shows a high effect on the expression of the differentiation function of cells.
以下、実施例に基づき本発明をさらに具体的に説明す
る。Hereinafter, the present invention will be described more specifically based on examples.
〔実施例１〕 基質の調製は、以下のように行つた。Example 1 Preparation of a substrate was performed as follows.
ヘパリン（ナカライテスク社）、κ−カラゲナン（和
光純薬社）、コンドロイチン硫酸Ｃ（ナカライテスク
社）、グリコーゲン（sigma社）、ラミナリン（sigma
社）をそれぞれ純水に溶解し、0.1％液を調製した。Heparin (Nacalai Tesque), κ-carrageenan (Wako Pure Chemical Industries), chondroitin sulfate C (Nacalai Tesque), glycogen (sigma), laminarin (sigma)
Was dissolved in pure water to prepare 0.1% solutions.
0.22μｍのフイルターで濾過後、35mmφデイツシユ
（住友ベークライト社.MS−10350）にコートした。クリ
ーンベンチ内で数時間静置後真空乾燥機を用い乾燥し
た。After filtration with a 0.22 μm filter, the resultant was coated on a 35 mmφ date (Sumitomo Bakelite, MS-10350). After leaving it to stand in a clean bench for several hours, it was dried using a vacuum dryer.
次に、内部電極型プラズマ重分装置（サムコエンジニ
アリング社.PD−10）を用いて低温プラズマ処理を行つ
た。Next, low-temperature plasma treatment was performed using an internal electrode type plasma heavy separator (SAMCO ENGINEERING, LTD. PD-10).
上記デイツシユを反応容器内に入れ0.02Torrまで減
圧、反応ガスである一酸化炭素ないしはアンモニアを導
入した。0.04〜0.05Torrに保ち、電圧を印加し、50Wで1
0分処理した。The above-mentioned dish was placed in a reaction vessel, and the pressure was reduced to 0.02 Torr, and carbon monoxide or ammonia as a reaction gas was introduced. Keep voltage between 0.04 and 0.05 Torr, apply voltage,
Treated for 0 minutes.
次に、クリーンベンチ内の殺菌灯により１〜３時間殺
菌した。この基質を用いた細胞培養は以下のように行つ
た。Next, it sterilized by a germicidal lamp in a clean bench for 1 to 3 hours. Cell culture using this substrate was performed as follows.
神経系細胞である副腎髄質褐色細胞腫PC12を培養し
た。牛胎児血清を10％、馬血清を５％添加したダルベツ
コ変法MEM培地（日水製薬社，以下DMEM）を用いて、３
×104個/mlの濃度に調製、上記デイツシユ各２枚に2ml
ずつ加え、37℃、５％CO2に調整したインキユベーター
内で一夜培養した。培地を除いた後血清無添加DMEMで１
回リンス後50ng/mlのNGF（タカラ酒造社）添加培地をDM
EMで調製したもので培地変換を行い、２日間培養した。
NGFの添加によりPC12は分化を行い神経突起を形成する
が、この神経突起の長さで３種類に分類し、それぞれを
カウントした。Neural cells, adrenal medulla pheochromocytoma PC12, were cultured. Using a Dulbecco's modified MEM medium (Nissui Pharmaceutical, hereinafter DMEM) supplemented with 10% fetal calf serum and 5% horse serum, 3
Adjust to a concentration of × 10 4 pieces / ml.
And cultured overnight at 37 ° C. in an incubator adjusted to 5% CO 2 . After removing the medium, add 1 serum-free DMEM.
After the first rinse, add 50 ng / ml NGF (Takara Shuzo) -added medium to DM
The medium was changed with the one prepared by EM and cultured for 2 days.
PC12 differentiates and forms neurites by the addition of NGF. The neurites were classified into three types according to their length, and each was counted.
その結果は表１に示すとおり、本発明により調製した
ヘパリン，コンドロイチン酸、κ−カラゲナンのスルホ
ン基含有多糖にプラズマ処理した基質は長い神経突起を
形成するものの比率が高く、高い分化機能発現の効果を
示すことが判明した。それに反し、スルホン基を持たな
い多糖、低温プラズマ処理を行なわない場合は、いずれ
も低い効果しか得られなかつた。The results are shown in Table 1. As shown in Table 1, the substrate treated with the heparin, chondroitic acid, and sulphonate-containing polysaccharide of κ-carrageenan prepared according to the present invention has a high ratio of those that form long neurites, and the effect of high differentiation function expression is high. It turned out to show. On the other hand, when the polysaccharide having no sulfone group and the low-temperature plasma treatment were not performed, only a low effect was obtained.
〔実施例２〕 ヘパリンκ−カラゲナン、コンドロイチン硫酸Ｃは実
施例１と同様に調製した。ゼラチン（Sigma社 生化学
用）、キトサン10C（カトキチ社）は、37℃に加温した
純水を用いて0.1％液を調製し、0.22μｍフイルターで
濾過した。 Example 2 Heparin κ-carrageenan and chondroitin sulfate C were prepared in the same manner as in Example 1. Gelatin (for Biochemistry, Sigma) and Chitosan 10C (Katokichi) were prepared in 0.1% solution using pure water heated at 37 ° C., and filtered with a 0.22 μm filter.
ヘパリン、κ−カラゲナン，コンドロイチン硫酸Ｃの
溶液とゼラチンあるいはキトサン10Cの溶液とそれぞれ
等量混合し、実施例１と同様に35mmφデイツシユにコー
トして一酸化炭素プラズマ処理を行つた。また、同様
に、PC12細胞を用いて評価した。その結果は、表２に示
すごとく、スルホン基を有する多糖とゼラチンないしは
キトサン10Cからなる組成物は、良好な分化機能発現の
効果を示すことが判明した。Equal amounts of a solution of heparin, kappa-carrageenan, and chondroitin sulfate C and a solution of gelatin or chitosan 10C were mixed in equal amounts, and coated on a 35 mm diameter date and treated with carbon monoxide plasma as in Example 1. Similarly, evaluation was performed using PC12 cells. The results, as shown in Table 2, revealed that a composition comprising a polysaccharide having a sulfone group and gelatin or chitosan 10C exhibited a good effect of expressing a differentiation function.
Claims (5)
理したことを特徴とする細胞培養用基質。1. A cell culture substrate obtained by subjecting a polysaccharide having a sulfone group to low-temperature plasma treatment.
る組成物を低温プラズマ処理したことを特徴とする細胞
培養用基質。2. A cell culture substrate obtained by subjecting a composition comprising a polysaccharide having a sulfone group and gelatin to low-temperature plasma treatment.
を有する多糖からなる組成物を低温プラズマ処理したこ
とを特徴とする細胞培養用基質。3. A cell culture substrate obtained by subjecting a composition comprising a polysaccharide having a sulfone group and a polysaccharide having an amino group derivative to low-temperature plasma treatment.
パラン硫酸、コンバロイチン硫酸、ケラタン硫酸、カラ
ゲナンから選択される請求項第（１）項ないし第（３）
項記載の細胞培養用基質。4. The polysaccharide having a sulfone group is selected from heparin, heparan sulfate, convaloitin sulfate, keratan sulfate, and carrageenan.
The substrate for cell culture according to the above item.
キトサン塩酸塩、ヒアルロン酸、コンドロイチン、テイ
クロン酸から選択される請求項第（３）項記載の細胞培
養用基質。5. The polysaccharide having an amino group derivative is chitin,
The cell culture substrate according to claim 3, wherein the substrate is selected from chitosan hydrochloride, hyaluronic acid, chondroitin, and teicuronic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1252061A JP2609549B2 (en) | 1989-09-29 | 1989-09-29 | Cell culture substrate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1252061A JP2609549B2 (en) | 1989-09-29 | 1989-09-29 | Cell culture substrate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03117483A JPH03117483A (en) | 1991-05-20 |
| JP2609549B2 true JP2609549B2 (en) | 1997-05-14 |
Family
ID=17232016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1252061A Expired - Lifetime JP2609549B2 (en) | 1989-09-29 | 1989-09-29 | Cell culture substrate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2609549B2 (en) |
-
1989
- 1989-09-29 JP JP1252061A patent/JP2609549B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03117483A (en) | 1991-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE3783643T2 (en) | ADHESIVE BIOMATERIALS FOR ADHAESION OF CELLS AND TISSUES. | |
| Chen et al. | Immobilization of chitosan gel with cross-linking reagent on PNIPAAm gel/PP nonwoven composites surface | |
| Li et al. | Culture of primary rat hepatocytes within porous chitosan scaffolds | |
| Kumar et al. | The combined influence of substrate elasticity and surface-grafted molecules on the ex vivo expansion of hematopoietic stem and progenitor cells | |
| AU614747B2 (en) | Cell culture of anchorage dependent cells, materials and products | |
| CA2373639A1 (en) | Adhesive n,o-carboxymethylchitosan coatings which inhibit attachment of substrate-dependent cells and proteins | |
| Zheng et al. | The behavior of MC3T3‐E1 cells on chitosan/poly‐L‐lysine composite films: Effect of nanotopography, surface chemistry, and wettability | |
| EP3561043A1 (en) | Cell culture substrate | |
| Madihally et al. | Maintenance of CD34 expression during proliferation of CD34+ cord blood cells on glycosaminoglycan surfaces | |
| De Bartolo et al. | Novel membranes and surface modification able to activate specific cellular responses | |
| JP2609549B2 (en) | Cell culture substrate | |
| JPH05260950A (en) | Collagen-coated cell culture apparatus and its production | |
| JP5500754B2 (en) | Cell culture substrate made of clay hydrogel | |
| JP2609559B2 (en) | Substrates used for culturing tissue cells | |
| JP4269256B2 (en) | Nerve cell culture substrate and method for producing the same | |
| JP2628536B2 (en) | Cell culture substrate | |
| JPH06277038A (en) | Cell culture medium | |
| JPH01500641A (en) | Immobilized serotonin and indole analogs | |
| DE68913602T2 (en) | Tissue immobilization and cell culture system and method for attaching biologically active parts to a substrate. | |
| JPH04330277A (en) | Culture substrate | |
| JPH05227944A (en) | Polylysine-coated tool for culturing cell and its production | |
| Shimbara et al. | Long-term culture of functional hepatocytes on chemically modified collagen gels | |
| Lim et al. | Enhanced culturing of adipose derived mesenchymal stem cells on surface modified polystyrene Petri dishes fabricated by plasma enhanced chemical vapor deposition system | |
| JPS6251982A (en) | Substrate for cultivating cell | |
| RU2328527C1 (en) | Microcarrier for growing substrate-dependent animal cells in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090213 Year of fee payment: 12 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090213 Year of fee payment: 12 |
|
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100213 Year of fee payment: 13 |
|
| EXPY | Cancellation because of completion of term | ||
| FPAY | Renewal fee payment (prs date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100213 Year of fee payment: 13 |