异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养
1Department of Chemical and Materials Engineering,National Central University, 2Department of Botany and Microbiology,King Saud University, 3Cathay Medical Research Institute,Cathay General Hospital, 4Graduate Institute of Systems Biology and Bioinformatics,National Central University, 5Department of Medical Microbiology and Parasitology,Universiti Putra Malaysia, 6Department of Internal Medicine,Taiwan Landseed Hospital, 7Department of Zoology,Bharathiar University, 8Thiruvalluvar University
Summary
提出了一种不同刚度的聚乙烯醇-共衣酸凝胶的制备方法, 并与无肽接枝, 研究了生物材料的硬度对其分化和增殖的影响。干细胞。水凝胶的刚度受交联时间的控制。
Abstract
一些研究人员研究了物理线索, 如生物材料的硬度对干细胞增殖和分化的影响。然而, 这些研究人员大多用聚丙烯酰胺水凝胶进行干细胞培养。因此, 它们的结果是有争议的, 因为这些结果可能来源于聚丙烯酰胺的具体特性, 而不是生物材料的物理提示 (刚度)。在这里, 我们描述了一个用于制备水凝胶的协议, 它不是以聚丙烯酰胺为基础的, 它可以培养各种干细胞, 包括人类胚胎干细胞和人类诱导的多能干细胞 (iPS) 细胞。从 bioinert 聚乙烯醇-共衣酸 (P IA) 制备了不同刚度的水凝胶, 通过改变交联时间来控制交联度的刚度。以细胞外基质为基础, 研究了肽的 P IA 水凝胶作为干细胞培养和分化的未来平台。本文详细介绍了羊水干细胞、脂肪源性干细胞、人 ES 细胞和人类 iPS 细胞的培养和传代。肽的 P IA 水凝胶表现出优异的性能, 这是由它们的刚度特性引起的。该协议报告了生物材料的合成, 他们的表面操作, 以及控制的刚度性质, 最后, 它们对干细胞的命运的影响, 利用异的自由文化条件。根据最近的研究, 这种改良的基板可以作为未来的平台, 支持和指导各种干细胞线的命运, 以不同的联系;进一步, 再生和恢复失去的器官或组织的功能。
Introduction
干细胞分化为特定细胞谱系的命运和干细胞的长期增殖, 特别是人类诱导的多能 (iPS) 细胞和人类胚胎干细胞, 已知受抑制剂、生长因子和/或培养培养基中的小生物活性分子。最近, 生物材料的物理线索, 特别是细胞培养生物材料的硬度, 已被认为是一个重要的因素, 引导干细胞增殖和分化的命运1,2, 3、4、5、6。因此, 一些研究人员已经开始研究干细胞的命运, 它们在水凝胶上培养, 分化, 主要是采用不同刚度的聚丙烯酰胺水凝胶。
生物材料的硬度可以控制局灶粘连, 细胞形态学, 细胞表型和干细胞黏附, 特别是在二维 (2 维) 培养1,2,3,5。干细胞对生物材料的机械传感通常是通过整合素受体的黏着性信号来控制的。NMMIIA, nonmuscle 肌球蛋白 IIA 与肌动蛋白细胞骨架的依赖性收缩作用在2维细胞培养系统中的干细胞 mechanosensing 过程中起着至关重要的角色3,4,5, 7,8,9,10,11。
恩和他的同事们提出了一个有趣的观点, 即成体干细胞, 如骨髓干细胞培养的细胞培养生物材料具有类似的刚性与特定组织, 往往分化成细胞起源于特定的组织5。在膨胀介质中, 培养的2维软质聚丙烯酰胺水凝胶 (与脑组织的硬度可比) 被自发诱导分化为早期神经元谱系, 而 bms 细胞在在2维聚丙烯酰胺水凝胶3,5中, 与肌肉或胶原骨组织相似的水凝胶分别被发现诱导分化为心肌细胞和成骨细胞的早期谱系。许多研究者研究了胶原蛋白固定化培养的聚丙烯酰胺水凝胶的干细胞命运. i12,13,14,15,16,17,18,19,20,21. 但是, 应该提到一些相互矛盾的报告1,18,22,23,24为恩建议的知名的想法存在et al.5这是因为恩的想法5仅是在聚丙烯酰胺水凝胶上开发的, 其结果来源于生物材料 (聚丙烯酰胺) 的特定特性, 而不仅仅是物理提示 (刚度)生物材料因此, 开发另一种类型的水凝胶是很重要的, 其中的刚性可以通过交联凝胶来控制。为此, 研制了 bioinert 水凝胶, 由聚乙烯醇-共衣康酸 (P IA) 与不同的刚度, 这是由交联度控制与改变交联时间25,26,27,28,29,30,31,32. 干细胞可以在 nonmodified 的 p-ia 水凝胶上培养, 以及与胞外基质 (ECMs) 和肽接枝的 p ia 水凝胶。在先前的研究25中, 从脐带血中培养出的人造血干细胞 (hHSCs) 在不同刚度值的 P IA 水凝胶中进行, 从3帕斯卡到30帕斯卡的储存模数, 其中纤维连接蛋白或肽来自纤维连接蛋白 (CS1, EILDVPST) 被嫁接到 P IA 水凝胶。在 CS1 或纤维连接蛋白接枝的 P IA 水凝胶中观察到 hHSCs 的高体外折叠扩张, 显示了从12帕斯卡到30帕的中间刚度不等的 25。
人类 ips 和 ES 细胞不能在传统的组织培养聚苯乙烯 (TCP) 菜肴上培养33,34 , 因为人类 es 和 ips 细胞需要对 ECMs (如受精或层粘连蛋白) 进行特定的绑定, 以保持其多在长期文化。因此, 设计并制备了具有最佳刚度特性的肽接枝型 P IA 水凝胶结构, 并在单链、单链、联合段、双链、节段、分支式链32。从 ECMs 的整合素和多糖结合域中选择了肽序列。受精肽与双链或关节段连接的 P IA 水凝胶, 其储存模量在大约25人民军, 支持人类 ES 和 iPS 细胞的长期培养, 超过12个通道在无异和化学定义的条件32。在水凝胶上的关节段和双链细胞黏附分子促进了人类 ES 和 iPS 细胞的增殖和多32。这里, 一个关于制备 P IA 水凝胶的协议 (以储存模数从10人民军到30帕, 这是测量在空气湿条件下) 接枝和没有肽或 ECMs 描述。如何培养和通过几个干细胞 (包括羊水干细胞, 脂肪来源干细胞, 人 ES 细胞, 人类 iPS 细胞) 显示。
Protocol
本研究的实验得到了台湾 Landseed 医院 (IRB-13-05) 和国立中央大学伦理委员会的批准。在本研究中, 所有的实验都是按照所有相关的和适用的政府和机构的指导方针和规定进行的。 1. 解决方案和媒体准备 高分子提纯 用乙醇洗涤 p ia 水解 > 96.5% 的羧酸基团净化 p ia。在500毫升的圆锥烧杯中将20克的 P IA 放入200毫升的乙醇中, 在磁力搅拌器上搅拌 24-30 h. ?…
Representative Results
采用肽 (oligoECM) 或不同弹性的 ecm 接枝的 P IA 水凝胶是通过以下的反应方案来制备的, 如图 1A所示, 使用不同类型的 oligoECM (图 1B)。水凝胶的弹性由应用的交联强度 (时间) (图 1C) 调节。采用受精衍生肽接枝的 P IA 水凝胶, 具有25.3 帕 (24 h 交联时间) 的贮存模量, 支持人类 iPS 和 ES 细胞长期培养超过10-20 通道。…
Discussion
为了长期扩展人类 ES 和 iPS 细胞, 在异自由条件下维持其多超过十通道, 以及对人类 AFS 细胞、广告细胞的培养, 建立了具有不同刚度的 p-ia-oligoECM 和 p-ia 型 ECM 水凝胶,和造血干细胞25,28,32。oligoECM 固定化的 p-IA 水凝胶是细胞培养材料研究细胞培养材料对不同类型的干细胞分化和增殖的命运以及原代细胞的影响的绝佳候选物。癌?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了部分支持的科技部, 台湾根据赠款编号 106-2119-m-008-003, 105-2119-m-008-006, 和 104-2221-e–008-107-MY3。这项研究也得到了台湾 Landseed 医院项目 (NCU-LSH-105-A-001) 的支持。还承认日本教育、文化、体育、科学和技术部的科研资助 (数字 15K06591)。a. 口想感谢沙特阿拉伯王国利雅得11451沙特国王大学国际科学伙伴计划 (ISPP-0062) 的研究生研究和研究副校长。
Materials
| GTPGPQGIAGQRGVV | PH Japan | none | Specification: Oligopeptide Abbreviation: Cyclic RGD, cRGD |
| GACRGDCLGA | PH Japan | none | Specification: Oligopeptide Abbreviation: FN1 |
| KGGAVTGRGDSPASS | PH Japan | none | Specification: Oligopeptide Abbreviation: CS1 |
| EILDVPST | PH Japan | none | Specification: Oligopeptide Abbreviation: VN1 |
| KGGPQVTRGDVFTMP | PH Japan | none | Specification: Oligopeptide Abbreviation: HBP1 |
| GKKQRFRHRNRKG | PH Japan | none | Specification: Oligopeptide Abbreviation: HBP2C |
| CGGGKKQRFRHRNRKG | PH Japan | none | Specification: Oligopeptide Abbreviation: VN1G |
| GGGGKGGPQVTRGDVFTMP | PH Japan | none | Specification: Oligopeptide Abbreviation: VN2C |
| GCGGKGGPQVTRGDVFTMP | PH Japan | none | Specification: Extracellular matrix Abbreviation: rVN |
| Vitronectin | Thermo Fisher scientific | A14700 | Specification: Extracellular matrix Abbreviation: FN |
| Fibronectin | Sigma-Aldrich | F2006 | Specification: Commercially available coating material Abbreviation: Synthemax II |
| Synthemax II | Corning | 3535 | Specification: Polymer |
| Polyvinylalcohol-co-itaconic acid | Japan VAM & Poval | AF-17 | Specification: Chemical |
| Glutaraldehyde | Sigma-Aldrich | G5882 | Specification: Chemical |
| Na2SO4 | Sigma-Aldrich | 239313 | Specification: Chemical |
| H2SO4 | Sigma-Aldrich | 339741 | Specification: Chemical Abbreviation: EDC |
| N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride | Sigma-Aldrich | E7750 | Specification: Chemical Abbreviation: NHS |
| N-hydroxysuccinimide | Sigma-Aldrich | 56480 | Specification: Chemical |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Specification: Chemical |
| Triton-X100 | Sigma-Aldrich | T8787 | Specification: Cell culture consumable |
| Cell scraper | Corning | 3008 | Specification: Cell culture consumable |
| Dispase II | Sigma-Aldrich | SI-D4693 | Specification: Cell culture medium |
| Essential 6 | Thermo Fisher scientific | A1516401 | Specification: Cell culture medium |
| Essential 8 | Thermo Fisher scientific | A1517001 | Specification: Cell culture medium |
| DMEM/F12 | Thermo Fisher scientific | 11330-032 | Specification: Cell culture medium |
| DMEM | Thermo Fisher scientific | 12800-017 | Specification: Cell culture medium |
| MCDB 201 | Sigma-Aldrich | M6770 | Specification: ES cell |
| Human ES cell | WiCell Research Institute, Inc.. | WA09 | Specification: iPS cell |
| Human iPS cell | Riken Cell Bank | HS0077 | Specification: 35 mm Abbreviation: TCP |
| TCP dish | Corning | 353001 | Specification: 60 mm Abbreviation: TCP |
| TCP dish | Corning | 353002 | Specification: 24 well dish |
| 24 well dish | Corning | 353047 | Specification: Blocking agent |
| Bovine serum albumin | Sigma-Aldrich | A8806 | Specification: Detection reagent |
| Alkaline phosphatase live stain | Thermo Fisher scientific | A14353 | Specification: Detection reagent |
| Hematoxylin & eosin | Sigma-Aldrich | 1.05175 | Specification: EB formation dish |
| 6-well ultralow attachment dish | Corning | 3471 | Specification: Coating material |
| gelatin | Sigma-Aldrich | G9391 | Specification: Coating material |
| Matrigel | Corning | 354230 | Specification: Mice |
| NOD-SCID mice | National Applied Research Laboratories | None | Specification: Serum |
| Fetal bovine serum | Biological Industries | 04-001-1A | Specification: Antibiotic |
| antimycotic antibiotic | Thermo Fisher scientific | 15240-062 | Specification: Antibody |
| Antibody for Nanog | Invitrogen | MA1-017 | Specification: Antibody |
| Antibody for SSEA4 | Abcam | ab16287 | Specification: Antibody |
| Antibody for OCT3/4 | Invitrogen | PA5-27438 | Specification: Antibody |
| Antibody for Sox2 | Invitrogen | 48-1400 | Specification: Antibody |
| Antibody for Smooth Muscle Actin | Invitrogen | PA5-19465 | Specification: Antibody |
| Antibody for AFP | Invitrogen | PA5-21004 | Specification: Antibody |
| Antibody GFAP | Invitrogen | MA5-15086 | Specification: Antibody |
| Alexa Fluor 555 – conjugated Goat anti-Mouse antibody | Invitrogen | A-21422 | Specification: Antibody |
| Alexa Fluor 488 – conjugated Goat anti-Rabbit antibody | Invitrogen | A-11008 | Specification: Antibody |
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Citar este artículo
Sung, T., Li, H., Higuchi, A., Ling, Q., Yang, J., Tseng, Y., Pan, C. P., Alarfaj, A. A., Munusamy, M. A., Kumar, S., Hsu, S., Murugan, K. Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions. J. Vis. Exp. (132), e57314, doi:10.3791/57314 (2018).