CN103805510A - 细胞培养装置、以及无血清细胞培养方法 - Google Patents
细胞培养装置、以及无血清细胞培养方法 Download PDFInfo
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Abstract
本发明提出细胞培养装置、以及无血清细胞培养方法。该细胞培养装置包含：基质，其中该基质具有表面；以及聚合物位于该表面上，其中该聚合物系由第一单体及第二单体经由聚合反应所形成，其中该第一单体具有公式(I)所示结构，且该第二单体具有公式(II)所示结构：公式(I)公式(II)其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3；R3系为氢、或甲基；及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH。
Description
技术领域
本发明系有关于一种细胞培养装置以及细胞培养方法，且特别是有关于一种无血清细胞培养装置及细胞培养方法。
背景技术
动物或人类细胞的体外培养，在传统研究上系使用含有动物血清的体外培养基。然而血清之使用除了会使产物回收纯化之困难度提升外，甚且易伴随感染性物质污染、以及批次间品质不易控制的严重问题。
对于以无血清培养基(serum free medium,SFM)培养贴附细胞时，通常会将细胞外基质(extracellular matrix,ECM)涂布于培养器皿上或加于培养基中，协助细胞贴附生长，未贴附的细胞则会走向凋亡(apoptosis)。细胞外基质(extracellular matrix,ECM)的主要成分为糖胺聚糖(glycosaminoglycan、GAG)、或更包含纤维状蛋白质(fibrous protein，例如：胶原(collagen)、层粘连蛋白(laminin)、纤连蛋白(fibronectin)、弹性蛋白(elastin))。举例来说，Invitrogen公司所制造贩售的StemPro MSC SFM即需要搭配含细胞外基质(例如CELLstartTM、由Invitrogen制造及贩售)涂布于培养皿上。然而，由于细胞外基质绝大部分为生物性蛋白质，因此生产成本高，且大多系由人类组织或血液中提取纯化而得，因此品质较不稳定。
基于上述，发展出可替代细胞外基质以促进贴附细胞生长的培养装置，避免细胞外基质的使用，实为目前细胞培养技术所期盼。
发明内容
本发明提出一种细胞培养装置，包含：基质，其中该基质具有表面；以及，聚合物位于该表面上。值得注意的是，该聚合物系由第一单体及第二单体经由聚合反应所形成，其中该第一单体具有公式(I)所示结构，且该第二单体具有公式(II)所示结构：
其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3；R3系为氢、或甲基；及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH。在一个实施方案中，所述聚合物是无轨共聚物。
本发明亦提供一种无血清细胞培养方法，包含：提供具有聚合物之基质；以及，将细胞置于该聚合物表面，以进行细胞培养。其中，该聚合物系由第一单体及第二单体经由聚合反应所形成，其中该第一单体具有公式(I)所示结构，且该第二单体具有公式(II)所示结构：
其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3；R3系为氢、或甲基；及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH。值得注意的是，在本发明所述之无血清细胞培养方法中，并没有使用血清(serum)或细胞贴附因子(cellattachment factor)。在一个实施方案中，所述聚合物是无轨共聚物。
为让本发明之上述和其它目的、特征、和优点能更明显易懂，下文特举出较佳实施例，并配合所附图式，作详细说明如下：
附图说明
图1系为剖面示意图，用以说明本发明一实施例所述之细胞培养装置。
图2a-2c系分别为实施例51、58、及其对照组所述之细胞培养装置培养3天后，在显微镜观察下所拍摄的照片。
图3a-3g系分别为实施例14、16、18、20、22、24、及其对照组所述之细胞培养装置培养3天后，在显微镜观察下所拍摄的照片。
图4a-4c系分别为实施例67、70、及其对照组所述之细胞培养装置培养3天后，在显微镜观察下所拍摄的照片。
图5a-5c系分别为实施例99、101、及其对照组所述之细胞培养装置培养3天后，在显微镜观察下所拍摄的照片。
【主要组件符号说明】
10~基质；
11~上表面；
12~聚合物层；以及
100~细胞培养装置。
具体实施方式
本发明系揭露一种细胞培养装置以及一种无血清细胞培养方法，可在没有血清(serum)或细胞贴附因子(cell attachment factor)存在的状况下，利用特定的聚合物促进细胞贴附及生长，降低细胞的培养成本，并改善品质稳定性。
根据本发明一实施例，请参照第1图，本发明所述的细胞培养装置100，包含基质(substratum)10，其中该基质10具有表面11。聚合物层12，系配置于该表面11。值得注意的是，由于本发明系使用特定的聚合物层12来促进细胞贴附及生长，因此本发明所使用的基质10并无限定，只要可作为该聚合物层之承载(support)即可，可为习知所使用的任何基质。该基质的材质可例如为玻璃、陶瓷、树脂、塑料、或是半导体材料，而其形状亦无限制，在此为简化图示，图中仅以一平整矩形表示。此外，该基质10的表面11可为平面、曲面、或其结合，而该表面11可为平整的表面、粗糙的表面、或是具有复数孔洞的表面。本发明所述之聚合物层12，系由聚合物所构成，其中该聚合物包含由第一单体及第二单体经由聚合反应所得之产物。在此，该第一单体可为具有公式(I)所示结构之化合物：
其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3。举例来说，该第一单体可包含甲基丙烯酸甲酯(methyl methacrylate、MMA)、甲基丙烯酸甲氧基乙酯(methoxy ethyl methacrylate、MEMA)、丙烯酸甲酯(methyl acrylate、MA)、丙烯酸乙酯(ethyl acrylate、EA)、或其组合；而该第二单体可为具有公式(II)所示结构之化合物：
其中，R3系为氢、或甲基；以及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH。举例来说，该第二单体可包含甲基丙烯酸(methacrylic acid、MA-H)、琥珀酸单[2-[(2-甲基-丙烯酰基)氧]乙基]酯(mono-2-(methacryloyloxy)ethyl succinate、MAES-H)、2-丙烯酸羧乙酯(2-carboxy ethyl acrylate、CEA)、单(2-丙烯酰乙基醚)琥珀酸(mono-(2-acryloyloxyethyl)succinate、AES-H)、丙烯酸(acrylic acid、A-H)、马来酸单-2-(甲基丙烯酰氧基)乙酯(mono-2-(methacryloyloxy)ethyl maleate、MAEM-H)、或其组合。根据本发明某些实施例，用来进行聚合反应的该第一单体及该第二单体之摩尔比可为1:9至9:1，例如1:9至8:2、1:9至7:3、2:8至9:1、3:7至9:1、或是2:8至8:2。上述由第一单体及第二单体经由聚合反应所得之聚合物可具有重量平均分子量为800,000至4,000,000，且该聚合物之分子量分布系数(dispersion index)为1至3。在一个实施方案中，所述聚合物是无轨共聚物。
举例来说，本发明所述之聚合物其制备方式可包含以下步骤：首先，将该第一单体、第二单体、热起始剂(thermoinitiator)偶氮二异丁腈(azobis(isobutyro)nitrile,AIBN)及溶剂，如二甲基甲酰胺(dimethylformamide,DMF)或异丙醇(isopropyl Alcohol,IPA)，置于反应管中，内置搅拌棒，并放入反应釜(Radleys Carousel reactor)中，将反应管充填氮气30分钟以去除氧气；接着，将反应管封好，反应釜加热至60℃并搅拌过夜(overnight)。然后待冷却至室温后，反应后形成之聚合物再沉淀于150mL溶剂(例如：去离子水、饱和盐溶液(saturated salt solution)或乙醚(diethyl ether))中，沉淀聚合物再以溶剂(例如：去离子水、饱和盐溶液(saturated salt solution)或乙醚(diethyl ether))清洗，于40℃真空烘箱中将聚合物干燥。
本发明所揭露之细胞培养装置，包括基质10，将上述之聚合物涂布于该基质表面11之上，得到该聚合物层12(聚合物在涂布前可预先溶于挥发性溶剂(例如：四氢呋喃(tetrahydrofuran、THF))。基质表面可以加工处理表面等离子体(surface plasma)，以活化表面使得聚合物层更佳贴附于基质上。需进行此表面活化步骤者，仅限于用在特定聚合物及特定基质。依下列步骤方法将聚合物涂布于该基质表面：首先准备含有聚合物的溶剂，即将聚合物溶解于适当的溶剂中。然后，将此溶剂均匀涂布于该基质表面，将聚合物溶剂留在基质表面作用，接下来将之干燥或去除多余的溶液。此外，该布聚合物层的基质可放入40℃烘箱，以确保去除残留的溶剂。
形成聚合物以及将聚合物形成于基质表面上的方法可为业界所常用的方法。举例来说，可以利用等离子体聚合技术(plasma polymerization)或是等离子体诱导接枝聚合技术(plasma induced graft polymerization)以单一步骤直接形成聚合物薄膜层于基质表面上。此外，有些共聚合物也可能可以由块状聚甲基丙烯酸甲酯(bulk poly(methyl methacrylate))、即商品名为帕斯佩有机玻璃(Perspex)或俗称压克力的材料)水解(hydrolysis)制备而得。
本发明亦提供一种无血清细胞培养方法，无需使用血清(serum)或细胞贴附因子(cell attachment factor)，即可完成贴附细胞的培养。根据本发明另一实施例，该无血清细胞培养方法可包含：提供具有聚合物之基质；以及，将细胞置于该聚合物表面，以进行细胞培养。其中该聚合物系由上述具有公式(I)之第一单体及具有公式(II)之第二单体经由聚合反应所得。根据本发明一实施例，在该聚合物表面披覆之细胞可为贴附细胞(adherent cells)，例如间质干细胞(mesenchymal stem cells)、或成纤维细胞(dermal fibroblasts)；间质干细胞可来自不同组织来源，例如骨髓间质干细胞(bone marrow mesenchymalstem cells)、脂肪干细胞(adipose tissue-derived stem cells)、华顿凝胶干细胞(Wharton’s jelly stem cells，又称之为脐带干细胞)。在一个实施方案中，所述聚合物是无轨共聚物。
传统以血清培养基或是添加有细胞外基质(extracellular matrix,ECM)之培养基所进行的细胞培养程序，具有易伴随感染性物质污染、价格昂贵、以及细胞培养批次间品质不易控制等缺点。由于本发明所述之细胞培养方法可在完全不使用的血清及细胞外基质的条件下进行细胞培养，且所使用之聚合物成本低廉，适合临床应用，因此可有效取代传统血清培养基或是添加细胞外基质的培养方式。
为了让本发明之上述和其它目的、特征、和优点能更明显易懂，下文特举数实施例及比较实施例，来说明本发明所述之含聚亚酰胺之膜层、以及蚀刻含聚亚酰胺之膜层的方法。
聚合物之制备
表1列出于本发明在制备聚合物中所使用之化合物其结构、名称及代表符号，以期使本发明所述之制备例能更为清楚：
表1
制备例1
首先，将该第一单体(MMA)、第二单体(MAES-H)、偶氮二异丁腈(azobis(isobutyro)nitrile,AIBN)、及二甲基甲酰胺(Dimethylformamide,DMF)溶剂，置于反应管中，内置搅拌棒。其中，该第一单体与该第二单体之摩尔比为7:3。接着，将反应管放入反应釜(radleys Carousel reactor)中，并将反应管充入氮气30分钟再封好。然后，将反应釜加热至60℃，再冷却至室温后搅拌过夜(overnight)。反应后形成之聚合物再沉淀于150mL的去离子水中，沉淀聚合物再以去离子水清洗。最后，经干燥后，得到聚合物(1)。
聚合物以胶体渗透层析仪(gel Permeation Chromatography,GPC)量测所得之聚合物(1)之数量平均分子量(number average molecular weight、Mn)、重量平均分子量(weight average molecular weight、Mw)、以及分子量分布(dispersion index、Mw/Mn)，结果如表2所示。
制备例2-30
如制备例1之相同方式进行，但依据表2所述来取代制备例1所使用之第一单体、第二单体、以及第一单体与该第二单体之摩尔比，得到聚合物(2)-(30)。
接着，以胶体渗透层析仪量测所得之聚合物(2)-(30)之数量平均分子量(number average molecular weight、Mn)、重量平均分子量(weight averagemolecular weight、Mw)、以及分子量分布(dispersion index、Mw/Mn)，结果如表2所示。
表2
细胞培养
实施例1
首先准备溶液，将制备例1所得之聚合物(1)溶解于四氢呋喃(tetrahydrofuran、THF)。接着，以旋转涂布(spin-coating)方式，于室温真空下将聚合物干燥形成于圆形盖玻片(直径为13mm)上，得到聚合物层。接着，将人类骨髓间质干细胞(human bone marrow mesenchymal stem cell、BMSC)以3000个细胞/cm2的密度种入(seeded)该聚合物层内，并以BD MosaicTMhMSC SF Medium(由BD Biosciences制造及贩售)作为培养液(cell culturemedium)进行培养。在培养3天后，以自动细胞计数器(ADAM Cell Counter，由Digital Bio制造及贩售)评估细胞数量，并与对照组(所有条件与实施例1相同，但盖玻片上未形成该聚合物膜层)比较，结果请参照表3。
实施例2-61
如实施例1之相同方式进行细胞培养，但实施例2-61依据表3所述来取代实施例1所使用之聚合物、以及所培养的细胞种类。在培养3天后，以自动细胞计数器(ADAM Cell Counter，由Digital Bio制造及贩售)评估细胞数量，结果请参照表3。
表3
备注：
BMSC：人类骨髓间质干细胞(human bone marrow mesenchymal stemcell)；
ADSC:人类脂肪干细胞(human adipose tissue derived stem cell)；
Wj cells:人类脐带华顿凝胶干细胞(human umbilical cord Wharton’s jellystem cell)；
Fibroblast:人类包皮成纤维细胞(human foreskin fibroblasts、Hs68)。
请参照第2a、及2b图，系分别为实施例51、及58所述之细胞培养装置培养3天后，以显微镜(放大倍率为40倍)观察下所拍摄的照片；此外，请参照第2c图系为实施例51、及58之对照组(直接在盖玻片(不具有本发明所述之聚合物膜层)上培养BMSC细胞，并以BD MosaicTM hMSC SF Medium作为培养液(cell culture medium))所述之细胞培养装置在培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片；此外，请参照第2d图系为实施例51、及58之正对照组(在盖玻片涂布BD MosaicTM hMSC SF Surface Coating上培养BMSC细胞，并以BD MosaicTM hMSC SF Medium作为培养液(cell culturemedium))所述之细胞培养装置在培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片。由第2a至2c图可知，细胞在实施例51、及58所述之细胞培养装置中可以存活，且数目增加；反观在对照组中，则观察到细胞数目明显较少，且发生细胞死亡的现象。
请参照第3a至3f图，系分别为实施例14、16、18、20、22、及24(聚合物皆由相同之第一单体MMA及第二单体MA-H合成，但第一单体及第二单体比例不同，分别为7:3,6:4,5:5,4:6,3:7及1:9)所述之细胞培养装置培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片；此外，请参照第3g图系为该等实施例之对照组(直接在盖玻片(不具有本发明所述之聚合物膜层)上培养ADSC细胞，并以BD MosaicTM hMSC SF Medium作为培养液(cell culturemedium))所述之细胞培养装置培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片。由第3a至3g图可知，细胞在实施例14、16、18、20、22、及24所述之细胞培养装置中可以存活，且数目增加；反观在对照组中，则观察到细胞数目明显较少，且发生细胞死亡的现象。
实施例62
首先准备溶液，将制备例1所得之聚合物(1)溶解于四氢呋喃(tetrahydrofuran、THF)。接着，以旋转涂布(spin-coating)方式，于室温真空下将聚合物干燥形成于圆形盖玻片(直径为13mm)上，得到聚合物层。接着，将人类骨髓间质干细胞(human bone marrow mesenchymal stem cell、BMSC)以3000个细胞/cm2的密度种入(seeded)该聚合物层内，并以MesenCult-XFBasal Medium(由Stem Cell Technologies制造及贩售)作为培养液(cell culturemedium)进行培养程序。在培养3天后，以自动细胞计数器(ADAM CellCounter，由Digital Bio制造及贩售)评估细胞数量，并与对照组(所有条件与实施例62相同，但盖玻片上未形成该聚合物层)比较，结果请参照表4。
实施例63-85
如实施例62之相同方式进行细胞培养，但实施例63-85依据表4所述来取代实施例62所使用之聚合物、以及所培养的细胞种类。在培养3天后，以自动细胞计数器(ADAM Cell Counter，由Digital Bio制造及贩售)评估细胞数量，结果请参照表4。
表4
请参照第4a、及4b图，系分别为实施例67、及70所述之细胞培养装置培养3天后，以显微镜(放大倍率为40倍)观察下所拍摄的照片；此外，请参照第4c图系为实施例67、及70之对照组(直接在盖玻片(不具有本发明所述之聚合物层)上培养成纤维细胞，并以MesenCult-XF Basal Medium作为培养液(cellculture medium))所述之细胞培养装置培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片。由第4a至4c图可知，细胞在实施例67、及70所述之细胞培养装置中可以存活，且数目增加；反观在对照组中，则观察到细胞数目明显较少，且发生细胞死亡的现象。
实施例86
首先准备溶液，将制备例2所得之聚合物(2)溶解于四氢呋喃(tetrahydrofuran、THF)。接着，以旋转涂布(spin-coating)方式，于室温真空下将聚合物干燥形成于圆形盖玻片(直径为13mm)上，得到聚合物层。接着，将人类脂肪干细胞(human adipose tissue derived stem cell、ADSC)以3000个细胞/cm2的密度种入(seeded)该聚合物膜层内，并以StemPro MSC SFM BasalMedium(由Invitrogen制造及贩售)作为培养液(cell culture medium)进行培养。在培养3天后，以自动细胞计数器(ADAM Cell Counter，由Digital Bio制造及贩售)评估细胞数量，并与对照组(所有条件与实施例86相同，但盖玻片上未形成该聚合物层)比较，结果请参照表5。
实施例87-102
如实施例86之相同方式进行细胞培养，但实施例87-102依据表5所述来取代实施例86所使用之聚合物、以及所培养的细胞种类。在培养3天后，以自动细胞计数器(ADAM Cell Counter，由Digital Bio制造及贩售)评估细胞数量，结果请参照表5。
表5
请参照第5a、及5b图，系分别为实施例99、及101所述之细胞培养装置培养3天后，以显微镜(放大倍率为40倍)观察下所拍摄的照片；此外，请参照第5c图系为实施例99、及101之对照组(直接在盖玻片(不具有本发明所述之聚合物层)上培养Wj细胞，并以StemPro MSC SFM Basal Medium作为培养液(cell culture medium))所述之细胞培养装置培养3天后，在显微镜(放大倍率为40倍)观察下所拍摄的照片。由第5a至5c图可知，细胞在实施例99、及101所述之细胞培养装置中可以存活，且数目增加；反观在对照组中，则观察到细胞数目明显较少，且发生细胞死亡的现象。
由表3、表4、及表5的结果可知，本发明所述的细胞培养装置及细胞培养方法，使用的特定聚合物，可在没有血清(serum)或细胞贴附因子(cellattachment factor)存在的状况下，促进贴附细胞贴附及生长，降低细胞的培养成本，并改善品质稳定性。
虽然本发明已以数个较佳实施例揭露如上，然其并非用以限定本发明，任何所属技术领域中具有通常知识者，在不脱离本发明之精神和范围内，当可作任意之更动与润饰，因此本发明之保护范围当视后附之申请专利范围所界定者为准。
Claims (12)
1.一种细胞培养装置，包含：
基质，其中该基质具有表面；以及
聚合物位于该表面上，其中该聚合物系由第一单体及第二单体经由聚合反应所形成，其中该第一单体具有公式(I)所示结构，且该第二单体具有公式(II)所示结构：
其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3；R3系为氢、或甲基；及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH。
2.如权利要求1所述之细胞培养装置，其中该第一单体及该第二单体之摩尔比为1:9至9:1。
3.如权利要求1所述之细胞培养装置，其中该第一单体系包含甲基丙烯酸甲酯、甲基丙烯酸甲氧基乙酯、丙烯酸甲酯、丙烯酸乙酯、或其组合。
4.如权利要求1所述之细胞培养装置，其中该第二单体系包含甲基丙烯酸、琥珀酸单[2-[(2-甲基-丙烯酰基)氧]乙基]酯、2-丙烯酸羧乙酯、单(2-丙烯酰乙基醚)琥珀酸、丙烯酸、马来酸单-2-(甲基丙烯酰氧基)乙酯、或其组合。
5.一种无血清细胞培养方法，包含：
提供具有聚合物之基质，其中该聚合物系由第一单体及第二单体经由聚合反应所形成，其中该第一单体具有公式(I)所示结构，且该第二单体具有公式(II)所示结构：
其中，R1系为氢、或甲基；R2系为甲基、乙基、或-CH2CH2OCH3；R3系为氢、或甲基；及R4系为氢、-CH2CH2OCOCHCHCOOH、-CH2CH2OCOCH2CH2COOH、或-CH2CH2COOH；以及
将细胞置于该聚合物表面，以进行细胞培养。
6.如权利要求5所述之无血清细胞培养方法，其中系该细胞包含贴附细胞。
7.如权利要求6所述之无血清细胞培养方法，其中该贴附细胞系为间质干细胞、或成纤维细胞。
8.如权利要求5所述之无血清细胞培养方法，其中在该无血清细胞培养方法中，并无使用血清或细胞贴附因子。
9.如权利要求8所述之无血清细胞培养方法，其中该细胞贴附因子系为细胞外基质。
10.如权利要求5所述之无血清细胞培养方法，其中该第一单体及该第二单体之摩尔比为1:9至9:1。
11.如权利要求5所述之无血清细胞培养方法，其中该第一单体系包含甲基丙烯酸甲酯、甲基丙烯酸甲氧基乙酯、丙烯酸甲酯、丙烯酸乙酯、或其组合。
12.如权利要求5所述之无血清细胞培养方法，其中该第二单体系包含甲基丙烯酸、琥珀酸单[2-[(2-甲基-丙烯酰基)氧]乙基]酯、2-丙烯酸羧乙酯、单(2-丙烯酰乙基醚)琥珀酸、丙烯酸、马来酸单-2-(甲基丙烯酰氧基)乙酯、或其组合。
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