JPH0553473B2 - - Google Patents
Info
- Publication number
- JPH0553473B2 JPH0553473B2 JP60077325A JP7732585A JPH0553473B2 JP H0553473 B2 JPH0553473 B2 JP H0553473B2 JP 60077325 A JP60077325 A JP 60077325A JP 7732585 A JP7732585 A JP 7732585A JP H0553473 B2 JPH0553473 B2 JP H0553473B2
- Authority
- JP
- Japan
- Prior art keywords
- glutamate
- cells
- benzyl
- amino acid
- copolymer
- 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
- 229920001577 copolymer Polymers 0.000 claims description 19
- 239000000463 material Substances 0.000 claims description 15
- DHQUQYYPAWHGAR-UHFFFAOYSA-N dibenzyl 2-aminopentanedioate Chemical compound C=1C=CC=CC=1COC(=O)C(N)CCC(=O)OCC1=CC=CC=C1 DHQUQYYPAWHGAR-UHFFFAOYSA-N 0.000 claims description 12
- 238000004113 cell culture Methods 0.000 claims description 11
- 238000009408 flooring Methods 0.000 claims description 4
- 238000007334 copolymerization reaction Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 17
- 235000001014 amino acid Nutrition 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 11
- 230000021164 cell adhesion Effects 0.000 description 8
- HFZKKJHBHCZXTQ-JTQLQIEISA-N (4s)-4-azaniumyl-5-oxo-5-phenylmethoxypentanoate Chemical compound OC(=O)CC[C@H](N)C(=O)OCC1=CC=CC=C1 HFZKKJHBHCZXTQ-JTQLQIEISA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 4
- 229920000193 polymethacrylate Polymers 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- SEWIYICDCVPBEW-BYPYZUCNSA-N L-glutamate methyl ester Chemical compound COC(=O)[C@@H](N)CCC(O)=O SEWIYICDCVPBEW-BYPYZUCNSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229920001519 homopolymer Polymers 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- YEJSPQZHMWGIGP-UHFFFAOYSA-N dl-glutamic acid dimethyl ester Natural products COC(=O)CCC(N)C(=O)OC YEJSPQZHMWGIGP-UHFFFAOYSA-N 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- SEWIYICDCVPBEW-UHFFFAOYSA-N methyl glutamate Chemical compound COC(=O)C(N)CCC(O)=O SEWIYICDCVPBEW-UHFFFAOYSA-N 0.000 description 2
- OJTJKAUNOLVMDX-LBPRGKRZSA-N (2s)-6-amino-2-(phenylmethoxycarbonylamino)hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)OCC1=CC=CC=C1 OJTJKAUNOLVMDX-LBPRGKRZSA-N 0.000 description 1
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 1
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical class NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000000944 Soxhlet extraction Methods 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 150000002306 glutamic acid derivatives Chemical class 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 150000002668 lysine derivatives Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920005553 polystyrene-acrylate Polymers 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- UQDJGEHQDNVPGU-UHFFFAOYSA-N serine phosphoethanolamine Chemical compound [NH3+]CCOP([O-])(=O)OCC([NH3+])C([O-])=O UQDJGEHQDNVPGU-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Description
(a) 発明の技術分野
本発明はグルタミン酸ベンジルを含むアミノ酸
共重合体を表面に有することにより、細胞が付着
しやすいことを特徴とする細胞培養用床材に関す
るものである。
細胞培養用床材とは、細胞を培養する際に細胞
を多量に付着させて増殖をはかるために使用する
ものである。一般に、動物細胞は浮遊状態では増
殖せず、固体表面に付着してのみ増殖する性質を
有している。したがつてそのような細胞を培養す
るためには細胞を多量に付着する固体、つまり床
材が必要となる。
(b) 従来技術の説明
従来、細胞培養用床材はポリスチレン、ポリメ
タクリレートなどの合成高分子材料を用いて作製
されているが、これらの材料を用いた細胞培養用
床材では細胞が付着しにくい。
(c) 発明の目的
本発明は上記の問題をグルタミン酸ベンジルを
共重合成分として含むアミノ酸共重合体を用いる
ことにより、細胞付着性の高い細胞培養用床材を
提供することを目的とする。
(d) 発明の構成
本発明者は細胞の付着しやすい性質を有する材
料について種々研究を重ねたところ、グルタミン
酸ベンジルを共重合成分として含むアミノ酸共重
合体は細胞を多量に付着させる性質を有してお
り、細胞培養用床材として好適であることを見い
出し、本発明を完成するに到つた。
即ち、本発明の細胞培養用床材は、グルタミン
酸ベンジルを共重合成分として含むアミノ酸共重
合体をシート状、繊維状、ビーズ状、中空糸状な
どの目的とする形状に成型して得るか、あるいは
あらかじめ他の材料で目的とする形状に成型した
後、その表面に本発明のグルタミン酸ベンジルを
含むアミノ酸共重合体を塗布して得る。
本発明のアミノ酸共重合体構成素材としてのグ
ルタミン酸ベンジル及びその共重合成分としての
アミノ酸はＤ体、Ｌ体、ラセミ体でもよく、他の
アミノ酸として、アラニン、グリシン、メチオニ
ン、バリン、グルタミン酸誘導体、アスパラギン
酸誘導体、リジン誘導体、オルニチン誘導体など
が用いられる。アミノ酸共重合体の分子量はその
皮膜が形成される程度であればよく、また共重合
体中のグルタミン酸ベンジルの含量はアミノ酸の
種類によつて変わるが、10モル％以上が好まし
い。
(e) 発明の実施例
次に本発明を実施例によりさらに詳細に説明す
る。
実施例 １
Ｌ−グルタミン酸メチルとＬ−グルタミン酸ベ
ンジルとの共重合体（グルタミン酸メチルとグル
タミン酸ベンジルとのモル比＝１：１）の１，２
−ジクロルエタン溶液をガラス板上に流延し、風
乾して皮膜（膜厚約0.05mm）を得た。この皮膜を
エタノールでソツクスレー抽出を行つた後、乾燥
した。この皮膜上に、人由来の上皮性細胞を含む
培養液（約10万個／ml）を接触させたまま、炭酸
ガス濃度５％、湿度100％、37℃の部屋に静置し
た。17時間後、皮膜をリン酸緩衝液でかるく洗浄
し、皮膜上に付着している細胞の量を核染色法に
より定量した。
比較のため、グルタミン酸メチル単独重合体皮
膜及び細胞培養用床材に使用されているポリメタ
クリレートを用いて、同様の細胞付着試験を行つ
た。
皮膜に付着した細胞の量を標準試料に付着した
細胞の量で割ることにより、細胞付着率を求め
た。その結果を第１表に示す。
(a) Technical Field of the Invention The present invention relates to a bed material for cell culture, which is characterized by having an amino acid copolymer containing benzyl glutamate on its surface, thereby making it easy for cells to adhere to it. A cell culture bed material is used for culturing cells in order to allow a large amount of cells to adhere thereto in order to increase their proliferation. In general, animal cells do not grow in suspension, but only grow when attached to solid surfaces. Therefore, in order to culture such cells, a solid material, ie, a bed material, to which a large amount of cells can adhere is required. (b) Description of conventional technology Conventionally, cell culture bedding materials have been made using synthetic polymer materials such as polystyrene and polymethacrylate, but cell culture bedding materials made of these materials do not allow cells to adhere to them. Hateful. (c) Object of the Invention The object of the present invention is to solve the above-mentioned problem by using an amino acid copolymer containing benzyl glutamate as a copolymer component, thereby providing a cell culture bed material with high cell adhesion. (d) Structure of the Invention The present inventor has conducted various studies on materials that have properties that allow cells to easily adhere to them, and has discovered that an amino acid copolymer containing benzyl glutamate as a copolymer component has the property of allowing a large amount of cells to adhere to it. The present inventors have discovered that the present invention is suitable as a flooring material for cell culture, and have completed the present invention. That is, the cell culture bed material of the present invention can be obtained by molding an amino acid copolymer containing benzyl glutamate as a copolymerization component into a desired shape such as a sheet, fiber, bead, or hollow fiber; It is obtained by first molding into a desired shape using another material, and then applying the amino acid copolymer containing benzyl glutamate of the present invention to the surface thereof. Benzyl glutamate as a constituent material of the amino acid copolymer of the present invention and the amino acid as its copolymerization component may be D-form, L-form, or racemic form, and other amino acids include alanine, glycine, methionine, valine, glutamic acid derivatives, and asparagine. Acid derivatives, lysine derivatives, ornithine derivatives, etc. are used. The molecular weight of the amino acid copolymer is sufficient as long as it forms a film, and the content of benzyl glutamate in the copolymer varies depending on the type of amino acid, but is preferably 10 mol % or more. (e) Examples of the invention Next, the present invention will be explained in more detail using examples. Example 1 1,2 of a copolymer of methyl L-glutamate and benzyl L-glutamate (molar ratio of methyl glutamate and benzyl glutamate = 1:1)
-The dichloroethane solution was cast on a glass plate and air-dried to obtain a film (film thickness: approximately 0.05 mm). This film was subjected to Soxhlet extraction with ethanol and then dried. A culture solution containing human-derived epithelial cells (approximately 100,000 cells/ml) was left in contact with this film in a room at 37°C with a carbon dioxide concentration of 5% and humidity of 100%. After 17 hours, the film was gently washed with phosphate buffer, and the amount of cells adhering to the film was quantified by nuclear staining. For comparison, a similar cell adhesion test was conducted using a methyl glutamate homopolymer film and polymethacrylate used in cell culture flooring. The cell attachment rate was determined by dividing the amount of cells attached to the film by the amount of cells attached to the standard sample. The results are shown in Table 1.
【表】
この表から、この共重合体は対照試料であるポ
リメタクリレートより多量の細胞を付着すること
が明らかである。
実施例 ２
Ｌ−グルタミン酸メチルとＬ−グルタミン酸ベ
ンジルとの共重合体の１，２−ジクロルエタン溶
液のかわりに、カルボベンゾキシ−Ｌ−リジンと
Ｌ−グルタミン酸ベンジルとの共重合体（カルボ
ベンゾキシリジンとグルタミン酸ベンジルとのモ
ル比＝７：３）のジオキサン溶液を用いた以外は
実施例１と同様にして皮膜を成型し、細胞付着実
験を行つた。結果を第２表に示す。TABLE From this table it is clear that this copolymer attaches more cells than the control sample polymethacrylate. Example 2 Instead of a 1,2-dichloroethane solution of the copolymer of methyl L-glutamate and benzyl L-glutamate, a copolymer of carbobenzoxy-L-lysine and benzyl L-glutamate (carbobenzoxylysine A film was formed in the same manner as in Example 1, except that a dioxane solution having a molar ratio of 7:3 and benzyl glutamate was used, and a cell adhesion experiment was conducted. The results are shown in Table 2.
【表】
この表から、この共重合体は対照試料であるポ
リメタクリレートより多量の細胞を付着すること
が明らかである。
実施例 ３
実施例２の共重合体のジオキサン溶液（濃度約
0.5％）中にガラスビーズ（直径約0.5mm）を浸漬
し、その後溶媒を除去、乾燥して、カルボベンゾ
キシリジンとグルタミン酸ベンジルとの共重合体
を表面に有するビーズ状細胞培養用床材を得た。
実施例 ４
実施例１において、Ｌ−グルタミン酸ベンジル
とロイシン（モル比＝30：70）を用いた以外は同
様にして実験を行つた。その結果、細胞付着率14
％の成績が得られた。また、ロイシン単独重合体
では、その細胞付着率は３％であつた。
実施例 ５
実施例１において、Ｌ−グルタミン酸ベンジル
とＬ−ヒドロキシプロピルグルタミン（モル比＝
68：32）を用いた以外は同様にして実験を行つ
た。その結果、細胞付着率26％の成績を得た。ま
た、Ｌ−ヒドロキシプロピルグルタミン単独重合
体では、その細胞付着率は０％であつた。
実施例 ６
実施例１において、Ｌ−グルタミン酸ベンジル
とＬ−グルタミン酸メチル（モル比＝71：29）を
用いた以外は同様にして実験を行つた。その結
果、細胞付着率98％の成績が得られた。
(f) 発明の効果
本発明は以上説明したように、細胞付着性の高
いことを必要とする細胞培養用床材において、グ
ルタミン酸ベンジルを含むアミノ酸共重合体を表
面に成型することにより細胞の付着量を増すこと
が可能である。TABLE From this table it is clear that this copolymer attaches more cells than the control sample polymethacrylate. Example 3 A solution of the copolymer of Example 2 in dioxane (concentration approx.
Glass beads (approximately 0.5 mm in diameter) are immersed in 0.5%), then the solvent is removed and dried to create a bead-shaped cell culture bed material with a copolymer of carbobenzoxylidine and benzyl glutamate on the surface. Obtained. Example 4 An experiment was conducted in the same manner as in Example 1, except that benzyl L-glutamate and leucine (molar ratio = 30:70) were used. As a result, the cell attachment rate was 14
% results were obtained. Furthermore, the cell adhesion rate of leucine homopolymer was 3%. Example 5 In Example 1, benzyl L-glutamate and L-hydroxypropylglutamine (molar ratio =
The experiment was conducted in the same manner except that 68:32) was used. As a result, a cell adhesion rate of 26% was obtained. Furthermore, in the case of L-hydroxypropylglutamine homopolymer, the cell adhesion rate was 0%. Example 6 An experiment was conducted in the same manner as in Example 1 except that benzyl L-glutamate and methyl L-glutamate (molar ratio = 71:29) were used. As a result, a cell attachment rate of 98% was obtained. (f) Effects of the Invention As explained above, the present invention improves cell adhesion by molding an amino acid copolymer containing benzyl glutamate on the surface of a cell culture flooring material that requires high cell adhesion. It is possible to increase the amount.
Claims (1)
むアミノ酸共重合体を表面に有する細胞培養用床
材。1. A cell culture flooring material having on its surface an amino acid copolymer containing benzyl glutamate as a copolymerization component.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60077325A JPS61234775A (en) | 1985-04-11 | 1985-04-11 | Bed material for cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60077325A JPS61234775A (en) | 1985-04-11 | 1985-04-11 | Bed material for cell culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61234775A JPS61234775A (en) | 1986-10-20 |
| JPH0553473B2 true JPH0553473B2 (en) | 1993-08-10 |
Family
ID=13630781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP60077325A Granted JPS61234775A (en) | 1985-04-11 | 1985-04-11 | Bed material for cell culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61234775A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59201849A (en) * | 1983-04-30 | 1984-11-15 | 工業技術院長 | Polyamino acid film composite body |
-
1985
- 1985-04-11 JP JP60077325A patent/JPS61234775A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59201849A (en) * | 1983-04-30 | 1984-11-15 | 工業技術院長 | Polyamino acid film composite body |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61234775A (en) | 1986-10-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EXPY | Cancellation because of completion of term |