JPS63196276A - Substrate for cell culture - Google Patents
Substrate for cell cultureInfo
- Publication number
- JPS63196276A JPS63196276A JP2900687A JP2900687A JPS63196276A JP S63196276 A JPS63196276 A JP S63196276A JP 2900687 A JP2900687 A JP 2900687A JP 2900687 A JP2900687 A JP 2900687A JP S63196276 A JPS63196276 A JP S63196276A
- Authority
- JP
- Japan
- Prior art keywords
- substrate
- cells
- culture
- cell culture
- cell
- 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.)
- Pending
Links
- 239000000758 substrate Substances 0.000 title claims abstract description 37
- 238000004113 cell culture Methods 0.000 title claims abstract description 27
- 229920000642 polymer Polymers 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 239000012510 hollow fiber Substances 0.000 claims abstract description 10
- 150000002632 lipids Chemical class 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 5
- 239000002131 composite material Substances 0.000 claims abstract 2
- 239000000463 material Substances 0.000 claims description 29
- 235000000346 sugar Nutrition 0.000 claims description 9
- 150000008163 sugars Chemical class 0.000 claims description 8
- 230000035755 proliferation Effects 0.000 abstract description 7
- 238000000926 separation method Methods 0.000 abstract description 7
- 229920001577 copolymer Polymers 0.000 abstract description 6
- 230000007774 longterm Effects 0.000 abstract description 5
- 239000002861 polymer material Substances 0.000 abstract description 3
- 230000002209 hydrophobic effect Effects 0.000 abstract description 2
- 150000001720 carbohydrates Chemical class 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 47
- 239000000243 solution Substances 0.000 description 26
- 108090000288 Glycoproteins Proteins 0.000 description 18
- 102000003886 Glycoproteins Human genes 0.000 description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- 239000011148 porous material Substances 0.000 description 7
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 6
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 6
- 210000004102 animal cell Anatomy 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 229920001400 block copolymer Polymers 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 102100026735 Coagulation factor VIII Human genes 0.000 description 3
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 229920000307 polymer substrate Polymers 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- -1 vaccines Substances 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 2
- GUUVPOWQJOLRAS-UHFFFAOYSA-N Diphenyl disulfide Chemical compound C=1C=CC=CC=1SSC1=CC=CC=C1 GUUVPOWQJOLRAS-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000013464 silicone adhesive Substances 0.000 description 2
- 239000003760 tallow Substances 0.000 description 2
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2,2'-azo-bis-isobutyronitrile Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000030275 Chondronectin Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001371 alpha-amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- OYQYHJRSHHYEIG-UHFFFAOYSA-N ethyl carbamate;urea Chemical compound NC(N)=O.CCOC(N)=O OYQYHJRSHHYEIG-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 150000002298 globosides Chemical class 0.000 description 1
- 229920000578 graft copolymer Polymers 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 108700020610 human chondronectin Proteins 0.000 description 1
- 102000043667 human chondronectin Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- IQPQWNKOIGAROB-UHFFFAOYSA-N isocyanate group Chemical group [N-]=C=O IQPQWNKOIGAROB-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical group FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〈産業上の利用分野〉
この発明は、細胞培養用基材に関する。さらに詳細には
、動物細胞を培養するために使用される細胞培養用基材
に関するものである。DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a substrate for cell culture. More specifically, the present invention relates to a cell culture substrate used for culturing animal cells.
〈従来の技術〉
近年、生物の細胞を培養し、その細胞の代謝活動により
を用な生理活性物質、例えば、ワクチン、ホルモン、イ
ンターフェロン等を生産する研究が活発に行われている
。<Prior Art> In recent years, research has been actively conducted to culture biological cells and produce physiologically active substances such as vaccines, hormones, interferons, etc. by the metabolic activity of the cells.
このような方法において、従来、接着性動物細胞の培養
は、ガラス、プラスチック製のシャーレ、試験管、培養
ビンなどを用いて行なわれできた。In such methods, adherent animal cells have conventionally been cultured using glass or plastic petri dishes, test tubes, culture bottles, and the like.
また、最近、マイクロキャリアや中空糸を培養用基材と
して用い、より高密度の培養や、長期の培養を行なう試
みがなされつつある。接着性動物細胞を培養周基村上に
接着させ、増殖させるには、該基材表面と細胞の接着性
が良好であることと共に接着した細胞の形態、配列が、
細胞の伸展、増殖に有効な形態になっていることが必要
である。Recently, attempts have been made to use microcarriers and hollow fibers as culture substrates to achieve higher-density culture and longer-term culture. In order to adhere and proliferate adherent animal cells on a cultured substrate, it is necessary to have good adhesion between the cells and the surface of the substrate, as well as the morphology and arrangement of the adhered cells.
It is necessary to have a form that is effective for cell expansion and proliferation.
しかしながら、従来から細胞培養用基材として用いられ
ている高分子材料は賦形性、耐久性に優れるものの、」
−記接着性等の点に関して不適当であり、高密度かつ長
期間の細胞培養を行なうことができず、いずれも十分な
成果を上げるに至っていない。However, although the polymer materials conventionally used as substrates for cell culture have excellent shapeability and durability,
- They are unsuitable in terms of adhesive properties, etc., and cannot perform high-density and long-term cell culture, and neither method has achieved sufficient results.
一方、ある種の糖、蛋白質、脂質およびこれらの複合化
合物は、細胞の接着に関与することが知られている。高
分子材料上にこれらの物質をコーティングして培養用基
材とする試みがあり、既にコラーゲンやその変性物であ
るゼラチンを塗布した培養用シャーレが市販されている
他、可溶性フィブロインの架橋体が積層された細胞培養
床（特開昭６１−５２２８０号公報参照）が提案されて
いる。On the other hand, certain sugars, proteins, lipids, and complex compounds thereof are known to be involved in cell adhesion. Attempts have been made to coat polymeric materials with these substances as culture substrates, and culture dishes coated with collagen or gelatin, a modified product of collagen, are already commercially available, as well as cross-linked soluble fibroin. A stacked cell culture bed (see JP-A-61-52280) has been proposed.
〈発明が解決しようとする問題点〉
しかしながら、上記の従来技術は、第１に高分子基材へ
の糖や蛋白質などの固定化が十分でなく容易に脱離して
しまうこと、第２に表面が均質の高分子基村上に糖や蛋
白質などを固定しているため、糖蛋白質等の高次構造が
変形し、不安定となり、細胞との接着性に劣ると共に接
着した細胞の伸展、増殖が十分でなく、高密度、長期間
の細胞培養ができないという問題点がある。<Problems to be solved by the invention> However, with the above-mentioned conventional technology, firstly, sugars, proteins, etc. are not sufficiently immobilized on the polymer base material and are easily detached, and secondly, sugars and proteins are easily desorbed from the surface. Since sugars and proteins are immobilized on a homogeneous polymeric base, the higher-order structure of the glycoprotein is deformed and becomes unstable, resulting in poor adhesion with cells and the spread and proliferation of adhered cells. There is a problem in that it is not sufficient and it is not possible to culture cells at high density and for a long period of time.
く目 的〉
この発明は上記問題点に鑑みてなされたものであり、細
胞との接着性に優れ、細胞の増殖と機能維持を行うこと
ができ、高密度、長期間の細胞培養を可能ならしめる細
胞培養用基材を提供することを目的とする。Purpose This invention has been made in view of the above-mentioned problems, and has excellent adhesion with cells, can proliferate cells and maintain their functions, and enables high-density, long-term cell culture. The purpose of the present invention is to provide a substrate for cell culture that retains moisture.
く問題点を解決するための手段および作用ン上記の問題
点を解決すべくなされた、この発明の細胞培養用基材は
、ミクロ相分離構造を表面に有する高分子基材の表面上
に、糖、蛋白質、脂質およびそれらの複合化合物（以下
、これらを糖蛋白質等と称する）のいずれか１種類以上
からなる層が形成されていることを特徴とするものであ
る。Means and Effects for Solving the Problems The cell culture substrate of the present invention, which was made to solve the above problems, has a polymer substrate having a microphase-separated structure on its surface. It is characterized by having a layer formed of one or more of sugars, proteins, lipids, and complex compounds thereof (hereinafter referred to as glycoproteins, etc.).
なお、上記高分子基材は、多孔質材料であるのが好まし
い。また、上記基材が中空糸であるものが好ましい。さ
らには、基材上に形成される糖蛋白質等からなる層は、
単分子積層されているのが好ましい。Note that the polymer base material is preferably a porous material. Further, it is preferable that the base material is a hollow fiber. Furthermore, the layer consisting of glycoprotein etc. formed on the base material is
It is preferable that monomolecular layers are formed.
上記の構成において、ミクロ相分離構造とは、多くのブ
ロックおよびグラフト共重合体に発現し、機能性、物性
等が異なる２以上の相がラメラ状または海島状に分布す
る構造であり、ミクロ相分離構造を有する′表面に吸着
した糖蛋白質等は、その配向性と分布が基材のミクロ相
分離構造を反映して制御され、その結果、糖蛋白質、糖
脂質等を表面に有する細胞との接着性が安定し、細胞の
伸展と増殖に好ましい効果をもたらす。従って、本発明
にかかるミクロ相分離構造を表面に有する高分子基材の
表面上に糖蛋白質等からなる層が形成された細胞培養用
基材は、細胞の安定な接着を促すと共に接着した細胞の
良好な伸展および増殖を可能にすることができる。特に
、糖蛋白質等が単分子積層された場合にあっては、その
配向性と分布がより確実に制御される結果、上記の効果
が一層顕著となる。In the above structure, the microphase separation structure is a structure that occurs in many block and graft copolymers, in which two or more phases with different functionality, physical properties, etc. are distributed in a lamellar or sea-island pattern. The orientation and distribution of glycoproteins, etc. adsorbed on surfaces with separated structures are controlled by reflecting the microphase separation structure of the substrate, and as a result, they are able to interact with cells that have glycoproteins, glycolipids, etc. on their surfaces. It has stable adhesive properties and has a favorable effect on cell spreading and proliferation. Therefore, the cell culture substrate in which a layer made of glycoprotein etc. is formed on the surface of a polymer substrate having a microphase separation structure according to the present invention can promote stable adhesion of cells and can allow good spreading and proliferation of. In particular, when glycoproteins and the like are monomolecularly stacked, their orientation and distribution are more reliably controlled, and as a result, the above effects become even more remarkable.
また、上記高分子基材が、多孔質材料であるときは、多
孔質材料の孔を通じて物質代謝が容易となり長期に亘り
細胞培養することができる。特に、前記高分子基材が中
空糸であるものは、中空部内や中空糸の外側に培養液等
を潅流することにより、中空糸上に細胞を高密度に育成
、増殖させることができる。Furthermore, when the polymer base material is a porous material, substance metabolism is facilitated through the pores of the porous material, and cells can be cultured for a long period of time. In particular, when the polymer base material is a hollow fiber, cells can be grown and multiplied at high density on the hollow fiber by perfusing a culture solution or the like into the hollow portion or outside of the hollow fiber.
以下、この発明をより詳細に説明する。This invention will be explained in more detail below.
この発明で使用される高分子基材は、少なくとも表面に
ミクロ相分離構造を有する材料が用いられ、特に親水性
部分と疎水性部分よりなる共重合体のミクロ相分離構造
が好ましい。このような材料としては、例えば、セグメ
ント化ポリエーテルウレタンウレア、２−ヒドロキシエ
チルメタクリレートとスチレンまたはジメチルシロキサ
ンとの共重合体、ポリ−α−アミノ酸とポリジメチルシ
ロキサンとの共重合体等を挙げることができる。The polymer base material used in the present invention is a material having a microphase-separated structure at least on its surface, and a microphase-separated structure of a copolymer consisting of a hydrophilic portion and a hydrophobic portion is particularly preferred. Such materials include, for example, segmented polyether urethaneurea, copolymers of 2-hydroxyethyl methacrylate and styrene or dimethylsiloxane, copolymers of poly-α-amino acids and polydimethylsiloxane, etc. Can be done.
ミクロ相分離構造は、これら各成分の構成比を変えるこ
とにより調整することができる。一方の成分が非常に多
くなると海島構造を、各成分の比率が近いとラメラ構造
をとるようになる。The microphase separation structure can be adjusted by changing the composition ratio of each of these components. If one component is very large, it will take on a sea-island structure, and if the ratios of each component are close, it will take on a lamellar structure.
この発明に適用される高分子基材は、上記のミクロ相分
離構造を有する重合体から直接形成された成形品やフィ
ルム、チューブ、中空糸、繊維、微粒子でもよいし、ま
たガラスや他の高分子材料により上記の形状に形成され
た成形品の表面に、これら共重合体を積層してなるもの
でもよい。基材の形状では、特に機能を維持しての長期
細胞培養を行うには、物質代謝を容易にする孔をもった
多孔性材料が有用であり、さらに高密度培養を行うには
、中空糸の形状が適している。中空糸としては、種々の
大きさのものが使用でき、例えば、内径５０〜１０００
７７１１程度のものが用いられる。The polymer base material applied to this invention may be a molded article, film, tube, hollow fiber, fiber, or fine particle formed directly from the polymer having the above-mentioned microphase-separated structure, or may be a glass or other polymer material. These copolymers may be laminated on the surface of a molded article formed into the above shape from a molecular material. Regarding the shape of the substrate, porous materials with pores that facilitate material metabolism are particularly useful for long-term cell culture while maintaining functionality, and hollow fiber materials are useful for high-density culture. The shape of is suitable. Hollow fibers of various sizes can be used, for example, those with an inner diameter of 50 to 1000
About 7711 is used.
また、この発明にかかる細胞培養用基材をマイクロキャ
リアー法のビーズ担体として使用する場合には、ビーズ
は１００〜３００μｍ程度の粒径のものが用いられる。Further, when the cell culture substrate according to the present invention is used as a bead carrier in a microcarrier method, beads having a particle size of about 100 to 300 μm are used.
この発明の細胞培養基材は、これらの高分子材料の表面
上に糖蛋白質等からなる層が形成された構造よりなり、
ここで用いる糖蛋白質等は、細胞と親和性があり、基材
と細胞の接着を促進するものであればいずれも用いるこ
とができ、例えば、ラクトース、ガラクトース等のオリ
ゴ糖、アルブミン等の蛋白質、リン脂質等の脂質、グロ
ボシド、ガングリオシド等の糖と脂質との複合体である
糖脂質、細胞質や血清中に含まれる脂質と蛋白質との複
合体であるリボ蛋白質、糖と蛋白質との複合体である糖
蛋白質等が挙げられ、特にオリゴ糖、コラーゲン、ゼラ
チン、フィブロネクチン、ラミニン、コンドロネクチン
、ビトロネクチン、フィブリン等の糖蛋白質が好適に用
いられ、これらは２種またはそれ以上組み合せて使用す
ることも有用である。基材上に糖蛋白質等からなる層を
形成する方法は、従来の技術がいずれも応用できる。The cell culture substrate of the present invention has a structure in which a layer made of glycoprotein etc. is formed on the surface of these polymeric materials,
The glycoproteins used here can be any as long as they have an affinity for cells and promote adhesion between the substrate and cells, such as oligosaccharides such as lactose and galactose, proteins such as albumin, Lipids such as phospholipids, glycolipids that are complexes of sugars and lipids such as globosides and gangliosides, riboproteins that are complexes of lipids and proteins contained in cytoplasm and serum, and complexes of sugars and proteins. Examples include certain glycoproteins, and glycoproteins such as oligosaccharides, collagen, gelatin, fibronectin, laminin, chondronectin, vitronectin, and fibrin are particularly preferably used, and it is also useful to use two or more of these in combination. It is. Any conventional technique can be applied to form a layer made of glycoprotein or the like on a base material.
一般には、ミクロ相分離構造を表面に宵する高分子基材
を、上記の糖蛋白質等の１種類または２種類以上を含有
する溶液に浸漬したり、該溶液を基材上に塗布した後、
乾燥することにより行われる。In general, a polymer base material having a microphase-separated structure on its surface is immersed in a solution containing one or more of the above-mentioned glycoproteins, or after coating the solution on the base material.
This is done by drying.
特に、これらが変性しにくい条件で乾燥するのが好まし
い。この際、特に単分子層で積層するのが好ましく、単
分子層の形成は、例えば、上記の基材を糖蛋白質等の稀
薄溶液と洗浄用緩衝溶液とに交互に浸漬することにより
なすことができる。In particular, it is preferable to dry under conditions that do not easily denature these materials. In this case, it is particularly preferable to form a monomolecular layer, and the monomolecular layer can be formed, for example, by alternately immersing the above-mentioned substrate in a dilute solution of glycoprotein or the like and a washing buffer solution. can.
この発明の細胞培養用基材は、種々の細胞の培養に使用
することができ、細胞の種類は特に限定されず生体由来
細胞、ハイブリドーマ−等が挙げられ、例えば、チャイ
ニーズハムスター肺由来細胞Ｖ−７９、ヒト子宮癌由来
細胞ＨｅＬａ、ヒト胎児肺由来細胞ＭＲＣ−５、ヒト肝
由来細胞Ｃｈａｎｇ Ｌｉｖｅｒ ｓヒト肺由来正二倍
体線維芽細胞ＩＲＣ−９０、ヒトリンパ腫由来ナマルバ
細胞等が例示される。The cell culture substrate of the present invention can be used for culturing various cells, and the type of cells is not particularly limited, and examples include living body-derived cells, hybridomas, etc. For example, Chinese hamster lung-derived cells V- 79, human uterine cancer-derived cells HeLa, human fetal lung-derived cells MRC-5, human liver-derived cells Chang Livers, human lung-derived eudiploid fibroblasts IRC-90, human lymphoma-derived Namalva cells, and the like.
また、この発明の細胞培養用基材を用いて動物細胞を培
養する場合、培養する細胞の種類に応じて種々の培養液
が用いられ、細胞の増殖に適した至適温度、ｐＨ等の条
件で培養が行なわれる。Furthermore, when culturing animal cells using the cell culture substrate of the present invention, various culture solutions are used depending on the type of cells to be cultured, and conditions such as optimal temperature and pH suitable for cell proliferation are used. Culture is carried out in
本発明の細胞培養用基材は、従来公知の種々のモジニー
ルにて、動物細胞の増殖に適用できる。The cell culture substrate of the present invention can be applied to the proliferation of animal cells using various conventionally known modules.
本発明の細胞培養基材として多孔質のチューブ状基材を
用いたモジュールの一例を、第１図および第２図に基づ
いて説明すると以下の通りである。An example of a module using a porous tubular base material as a cell culture base material of the present invention will be described below based on FIGS. 1 and 2.
第１図に示す細胞培養器（１）は、所定数集束された多
孔質チューブ状基材（２）が、細胞懸濁液を注入するた
めの孔（４）を有するポリカーボネート等からなる円筒
状容器（３）に装填されていると共に、上記基材■の両
端部がシリコーン接着剤（５）等により接着固定されて
いる。また、上記孔（４）は、細菌等で培養器（１）等
が汚染されるのを防止するため、フィルタ付きの蓋（４
ａ）で被冠されている。上記孔（４）より注入されて前
記基材（２）に接着した細胞を増殖させるため、上記円
筒状容器（３）の両端部には、培養液８）を供給する供
給口（７ａ）を備えたキャップ（６ａ）と、培養液［Ｆ
］）を排出する排出口（７ｂ）を備えたキャップ（６ｂ
）とがそれぞれ装着されており、培養器（１）内の前記
基材■に培養液［Ｆ］）を移送できるようにしている。The cell culture device (1) shown in Fig. 1 has a porous tubular base material (2) in which a predetermined number of cells are bundled, and a cylindrical shape made of polycarbonate or the like having holes (4) for injecting a cell suspension. It is loaded into a container (3), and both ends of the base material (1) are adhesively fixed with a silicone adhesive (5) or the like. In addition, the hole (4) is equipped with a lid (4) with a filter to prevent the incubator (1) etc. from being contaminated with bacteria etc.
It is crowned by a). In order to proliferate the cells injected through the hole (4) and adhered to the substrate (2), supply ports (7a) for supplying the culture solution 8) are provided at both ends of the cylindrical container (3). The provided cap (6a) and the culture solution [F
]) with a discharge port (7b) for discharging the cap (6b
) are attached to each of them, so that the culture solution [F]) can be transferred to the substrate (2) in the incubator (1).
上記の培養器（１）は、例えば、第２図に示されるシス
テムで用いられる。すなわち、新鮮な培養液［Ｆ］）を
培養器（１）に供給するため、上記培養器（１）の供給
口（７ａ）には、中間にポンプ００）が配されたパイプ
（１２ａ）が接、続されており、培養液容器（９）に収
容された新鮮な培養液口）を上記バイブ（１２ａ）を経
て上記培養器（１）に供給している。また、培養器（１
）を通過した培養液Ｓ）は、培養器（１）の排出口（７
ｂ）に接続されたパイプ（１２ｂ）を経て前記培養液容
器（９）に排出され、培養液（８）が上記システムを循
環するように構成されている。なお、上記培養器（１）
等は、インキュベータ（１１）内に配されている。The above-mentioned incubator (1) is used, for example, in the system shown in FIG. That is, in order to supply fresh culture solution [F]) to the incubator (1), a pipe (12a) with a pump 00) disposed in the middle is connected to the supply port (7a) of the incubator (1). A fresh culture solution (inlet) contained in a culture solution container (9) is supplied to the incubator (1) via the vibrator (12a). In addition, an incubator (1
) The culture solution S) that has passed through the incubator (1) is
b) is discharged into the culture liquid container (9) via a pipe (12b) connected to the culture medium (8), so that the culture liquid (8) circulates through the system. In addition, the above incubator (1)
etc. are placed in the incubator (11).
上記の培養器（１）およびシステムを用いて細胞を増殖
させるには、上記孔（４）から細胞懸濁液を注入して細
胞を前記基材（２）上に接着させると共に、前記孔（４
）をフィルタ付きのＭ （４ａ）で被冠し、所定の培養
条件の下、培養液容器（９）に収容された培養液［Ｆ］
）を前記バイブ（１２ａ） （１２ｂ）を通じて培養器
（１）内を所定時間循環させることにより行なわれる。To proliferate cells using the culture vessel (1) and system described above, a cell suspension is injected through the hole (4) to allow the cells to adhere to the substrate (2), and the hole ( 4
) was covered with M (4a) with a filter, and the culture solution [F] was placed in the culture solution container (9) under predetermined culture conditions.
) is circulated in the incubator (1) for a predetermined period of time through the vibrators (12a) and (12b).
〈実施例〉
以下、実施例に基づいて、この発明をより詳細に説明す
る。<Examples> Hereinafter, the present invention will be described in more detail based on Examples.
実施例１
片末端にアミノ基を有する２−ヒドロキシエチルメタク
リレートオリゴマーを、α、α″−アゾビスイソブチロ
ニトリル、２−アミノエタンチオールおよび２−ヒドロ
キシエチルメタクリレート（以下、ＨＥＭＡと称する）
のジメチルホルムアミド溶液を６０℃に加熱し、重合さ
せて合成した。Example 1 A 2-hydroxyethyl methacrylate oligomer having an amino group at one end was mixed with α, α″-azobisisobutyronitrile, 2-aminoethanethiol and 2-hydroxyethyl methacrylate (hereinafter referred to as HEMA).
The dimethylformamide solution of was heated to 60°C and polymerized to synthesize it.
また両端にイソシアナート基を有するスチレンオリゴマ
ーを、ｐ、ｐ−−ジイソシアネートジフェニルジスルフ
ィドとスチレン（以下、Ｓｔと称する）の光重合で合成
した。この２つのオリゴマーをジメチルホルムアミド中
で０℃、２４時間反応させ、ＨＥＭＡ−８ｔ−Ｉ（ＥＭ
Ａ型のブロック共重合体を得た。ＨＥＭＡモル分率０．
６でラメラ状のミクロ相分離構造を示すブロック共重合
体となった。このブロック共重合体をジメチルホルムア
ミドに溶解して１ｗｔ％溶液を調整後、表面処理された
ポリスチレンシャーレにコーティングし、４０℃で乾燥
した。このシャーレを紫外線滅菌後、無菌のコラーゲン
溶液（タイプ１１濃度０．１％）に１秒間接触させ、直
ちにリン酸緩衝溶液で洗浄し、室温で乾燥させた。これ
らの操作は、全て無菌のクリーンベンチ内で行なった。Furthermore, a styrene oligomer having isocyanate groups at both ends was synthesized by photopolymerization of p, p--diisocyanate diphenyl disulfide and styrene (hereinafter referred to as St). These two oligomers were reacted in dimethylformamide at 0°C for 24 hours, and HEMA-8t-I (EM
A type A block copolymer was obtained. HEMA mole fraction 0.
No. 6 resulted in a block copolymer exhibiting a lamellar microphase-separated structure. This block copolymer was dissolved in dimethylformamide to prepare a 1 wt % solution, which was then coated on a surface-treated polystyrene Petri dish and dried at 40°C. After sterilizing this petri dish with ultraviolet rays, it was brought into contact with a sterile collagen solution (type 11 concentration: 0.1%) for 1 second, immediately washed with a phosphate buffer solution, and dried at room temperature. All of these operations were performed in a sterile clean bench.
次に、このシャーレにチャイニーズハムスター肺由来細
胞（Ｖ−７９細胞）を培養液１１１当たりＩ Ｘ １０
４個播種し培養した。培養液は、１０重量％牛脂０児血
清を含むイーグルＭＥＭ培地を用い、５％炭酸ガス、９
５％空気雰囲気、温度３７℃の環境下７日間の培養を行
なった。培養後、細胞数は、培養液１１１当たり平均５
８１０６個となり、良好な増殖が観察された。Next, Chinese hamster lung-derived cells (V-79 cells) were added to this petri dish at a concentration of I×10 cells per 111 hours of culture solution.
Four seeds were sown and cultured. The culture solution used was Eagle's MEM medium containing 10% by weight beef tallow serum, 5% carbon dioxide gas, 9%
Culture was carried out for 7 days in a 5% air atmosphere at a temperature of 37°C. After culturing, the number of cells is on average 5 per 111 culture fluids.
The number of cells was 8106, and good growth was observed.
実施例２
実施例１と同じ手順に従い、ＨＥＭＡモル分率０．７の
ＨＥＭＡ−３ｔ−ＨＥＭＡ型のブロック共重合体を得た
。この共重合体をジメチルホルムアミドに溶解して１ｆ
ｆｉ量％溶液を調整した後、四フッ化エチレン樹脂から
なる多孔質チューブ（住人電気工業■製、外径２ ｍｍ
φ、内径ＩＩＩＩＩｌφ、気孔率５２％、平均孔径０，
３μＭ）に含浸させ、４０℃で乾燥した。ついで、この
チューブにフィブロネクチン（シグマ社製、牛血清より
採取）のリン酸緩衝溶液（濃度０ 、 １ ｍｇ ／
ｙｌ ）を塗布し、室温で乾燥させた。Example 2 According to the same procedure as in Example 1, a HEMA-3t-HEMA type block copolymer with a HEMA molar fraction of 0.7 was obtained. This copolymer was dissolved in dimethylformamide and 1f
After adjusting the fi amount % solution, a porous tube made of tetrafluoroethylene resin (manufactured by Jumi Electric Industry ■, outer diameter 2 mm) was prepared.
φ, inner diameter IIIIIIlφ, porosity 52%, average pore diameter 0,
3 μM) and dried at 40°C. Next, a phosphate buffered solution (concentration 0, 1 mg /
yl) was applied and dried at room temperature.
このチューブ１００本を集束し、第１図に示す培養容器
（１）を用いて細胞培養を行なった。すなわち、集束し
たチューブ状基材（２）をポリカーボネート製の円筒状
容器Ｇ）に充填し、端部をシリコーン接着剤（５）を用
いて接着固定した後、円筒の軸に垂直に切断することで
、チューブ状基材（２）の端部が全て露出した形の培養
器（１）を得た。These 100 tubes were collected and cells were cultured using the culture container (1) shown in FIG. That is, the bundled tubular base material (2) is filled into a polycarbonate cylindrical container G), the end portion is adhesively fixed using a silicone adhesive (5), and then cut perpendicularly to the axis of the cylinder. Thus, an incubator (1) in which the ends of the tubular base material (2) were completely exposed was obtained.
ついで、この両端部に培養液供給用の供給口（７ａ）が
ついたキャップ（６ａ）および排出口（７ｂ）がついた
キャップ（６ｂ）を装着し、外部から培養器（１）内部
のチューブ状基材（２）内室に培養液（８）を移送でき
る形とし、第２図に示すシステムと接続して、ハムスタ
ー仔腎由来細胞（ＢＨＫ−２１）の培養を行なった。培
養器（１）はあらかじめエチレンオキサイドでガス滅菌
し、殺菌した水、次いで培養液（８）でフラッシングを
行なった。細胞懸濁液（細胞数２．１Ｘ１０４個／１り
は、第１図に示す孔（４）より注入し、培養条件は、イ
ーグルＭ Ｅ Ｍ培地に１０％牛脂児血清を添加したも
のを用い、５％炭酸ガスを含む空気雰囲気中、３７℃で
インキュベイトして１５日間培養した。細胞はチューブ
の周辺に肉眼でも確認できるほど増殖しており、トリプ
シン−ＥＤＴＡ溶液で細胞を分離し細胞数を計算したと
ころ８．２Ｘ１０５個／１１ｉであった。Next, a cap (6a) with a supply port (7a) for supplying culture solution and a cap (6b) with a discharge port (7b) are attached to both ends, and the tube inside the culture vessel (1) is inserted from the outside. The substrate (2) was shaped so that the culture solution (8) could be transferred into the inner chamber, and was connected to the system shown in FIG. 2 to culture cells derived from baby hamster kidney (BHK-21). The culture vessel (1) was gas sterilized with ethylene oxide in advance and flushed with sterilized water and then with the culture solution (8). The cell suspension (2.1 x 104 cells per cell) was injected through the hole (4) shown in Figure 1, and the culture conditions were Eagle's MEM medium supplemented with 10% tallow serum. The cells were incubated and cultured for 15 days at 37°C in an air atmosphere containing 5% carbon dioxide.Cells had proliferated around the tube to the extent that they could be seen with the naked eye, and the cells were separated with a trypsin-EDTA solution to determine the cell number. When calculated, it was 8.2×105 pieces/11i.
〈発明の効果〉
以上のように、この発明の細胞培養用基材によれば、ミ
クロ相分離構造を表面に有する高分子基材の表面上に、
糖蛋白質等からなる層が形成されているので、糖蛋白質
等を基材上に強固かつその高次構造等を維持した安定な
形態で固定化することができる。さらに、糖蛋白質等は
、細胞との親和性に優れ、接着性を高めることができる
と共に細胞を伸展、増殖に適した形態、配列で接着させ
ることができるので、高密度かつ長期間の細胞培養が可
能となる。特に、糖蛋白質等からなる単分子層が形成さ
れた基材にあっては、その配向性と分布がより確実に制
御されるので、細胞との接着性並びに細胞の伸展性およ
び増殖性を一層高めることができるという特有の効果を
奏する。従って、この発明の細胞培養用基材は、動物細
胞の培養によるホルモン等の有用物の生産システムに利
用できる他、例えばインスリン産生細胞を基材表面に接
着、培養することにより人工膵臓が形成できるように人
工臓器の構築に利用できる。<Effects of the Invention> As described above, according to the cell culture substrate of the present invention, on the surface of the polymer substrate having a microphase separation structure on the surface,
Since the layer made of glycoprotein etc. is formed, the glycoprotein etc. can be firmly immobilized on the substrate in a stable form that maintains its higher-order structure. Furthermore, glycoproteins have excellent affinity with cells and can increase adhesion, as well as allow cells to adhere in a form and arrangement suitable for expansion and proliferation, allowing for high-density and long-term cell culture. becomes possible. In particular, in the case of substrates on which a monomolecular layer consisting of glycoproteins, etc. is formed, the orientation and distribution can be controlled more reliably, which further improves adhesion with cells, as well as cell spreadability and proliferation. It has the unique effect of being able to increase Therefore, the cell culture substrate of the present invention can be used in a system for producing useful products such as hormones by culturing animal cells, and can also form an artificial pancreas by, for example, attaching and culturing insulin-producing cells to the surface of the substrate. It can be used to construct artificial organs.
第１図は本発明の細胞培養用基材を用いた培養器の一例
を示す断面図であり
第２図はこの培養器を用いたシステムを説明するための
フロチャートである。
（１）・・・培養器、Ｃ）・・・チューブ状基材、（３
）・・・円筒状容器、（４）・・・孔、（８）・・・培
養液。FIG. 1 is a sectional view showing an example of a culture vessel using the cell culture substrate of the present invention, and FIG. 2 is a flowchart for explaining a system using this culture vessel. (1)...Incubator, C)...Tubular base material, (3
)... Cylindrical container, (4)... Hole, (8)... Culture solution.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2900687A JPS63196276A (en) | 1987-02-10 | 1987-02-10 | Substrate for cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2900687A JPS63196276A (en) | 1987-02-10 | 1987-02-10 | Substrate for cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63196276A true JPS63196276A (en) | 1988-08-15 |
Family
ID=12264317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2900687A Pending JPS63196276A (en) | 1987-02-10 | 1987-02-10 | Substrate for cell culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63196276A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02142468A (en) * | 1988-11-22 | 1990-05-31 | Bio Material Kenkyusho:Kk | Cell culture |
-
1987
- 1987-02-10 JP JP2900687A patent/JPS63196276A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02142468A (en) * | 1988-11-22 | 1990-05-31 | Bio Material Kenkyusho:Kk | Cell culture |
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