重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力
Summary
提出了大孔载体的细胞扩展协议及其在灌注生物反应器中的传递系统, 以细胞组织基质为种子。我们还包括不同的技术, 以确定细胞增殖和细胞在载体培养的生存能力。此外, 我们还展示了生物反应器培养后细胞的功能。
Abstract
组织工程学是一个有前途的领域, 重点是开发解决方案, 以增加对组织和器官的移植目的的需求。生成这种组织的过程是复杂的, 包括特定细胞类型、支架和物理或生物化学刺激的适当组合, 以指导细胞生长和分化。载体代表一个吸引人的工具来扩展三维 (3D) 微环境中的细胞, 因为它们提供了更高的表面体积比, 并且与传统的二维方法相比更接近于在体内的情况。血管系统, 提供氧气和养分的细胞和确保废物清除, 构成一个重要的积木时, 生成工程组织。事实上, 大多数的结构在植入后由于缺乏血管支持而失败。在这项研究中, 我们提出了一个协议的内皮细胞扩展的重组胶原基载体在动态条件下, 在微调瓶和生物反应器, 我们解释如何确定在这个设置细胞的可行性和功能。此外, 我们提出了一种细胞分娩的方法, 不需要额外的剥离步骤。此外, 我们还提供了一种在细胞生物基质上评价灌注生物反应器中细胞的血管潜能的策略。我们认为, 使用所提出的方法可能会导致开发新的细胞为基础的治疗组织工程的广泛应用在临床实践。
Introduction
组织工程应用中的一个普遍问题是在需要的位置上产生一个具有正确的分化表型的高细胞质量。在1967年开始应用载体来解决这一问题, 在诸如骨科组织工程等领域, 对于大规模的皮肤、骨骼、软骨和肌腱的产生具有越来越重要的意义1。它们允许通过在微尺度的三维 (3D) 基板上扩展细胞, 以类似于悬浮培养物的方式来处理附着的区域性2 。从而细胞体验均匀的营养素供应和细胞矩阵互作用导致更好的维护在体内3,4差异, 这通常在2D 方法5中随时间而丢失。一个更高的表面体积比-最终导致更高的细胞产量6,7, 与静态系统相比, 更高的气体和养分交换率8, 有可能调节并将文化置于物理刺激9, 并且扩展进程7的扩展潜力是进一步的优势。直径、密度、孔隙度、表面电荷和附着力等几个特性10,11区分了不同的商业可用的微型和宏载体。然而, 其中一个主要优势是他们的交付潜力, 作为 microtissues 的网站缺陷或需求。
对于载体技术在骨组织工程中的应用, 我们在以前的报告12中说明了一种新的载体类型的生产, 它是由重组的 i 型胶原肽 (RCP, 在商业上可作为 Cellnest)。这一新的载体允许符合 GMP 的支架和细胞生产, 根据需要的细胞分娩在临床情况。在这种情况下, 通过适当选择合适的交联策略来调整支架的稳定性、降解率和表面特性, 可以使技术适应选定的应用、细胞类型的兴趣或目标组织13。特别是, 这个载体作为一个可注射的治疗应用的细胞递送系统的潜在就业14使他们特别有趣的临床设置。
因此, 本文对人骨髓间充质基质细胞 (hBMSCs) 和人真皮微血管内皮细胞 (HDMECs) 的分离和扩展的培养程序进行了阐述, 对 i. 型胶原重组多肽基载体, 及其在临床设置分娩的准备。此外, 我们还描述了用于在移植后维持细胞活力的附加协议。
植入后的细胞活力实际上强烈依赖于血管化的15,16,17, 这保证了氧气和营养素的交换, 并促进了废物的去除。生物反应器是克服组织工程中血管化挑战和维持细胞活力的一种方法, 通过对培养基进行灌注, 从而提供氧气和营养素18。在这里, 我们演示了一个体外方法, 以评估从 RCP 载体微血管内皮细胞的迁移能力, 以博美和他们的能力, 以促进de 从头血管和血管生成。这博美是猪空肠的细胞段, 称为 BioVaSc (生物血管支架), 富含胶原蛋白和弹性蛋白, 并与保存血管结构, 其中包括喂养动脉和引流静脉19已已应用于植入问题20。
Protocol
hBMSCs 是骨关节炎患者股骨头置换术后股骨头的分离。该程序是在 Wuerzburg 大学地方伦理委员会的批准和病人的知情同意下进行的。原发微血管内皮细胞从幼年捐献者的包皮活检中分离出来。他们的法律代表以书面形式提供了充分的知情同意。这项研究得到了 Wuerzburg 大学地方伦理委员会的批准 (投票 182/10)。 1. hBMSCs 和 HDMECs 的隔离 人骨髓间充质细胞 (hBMSCs) 用无菌…
Representative Results
如图 1A所示, 在经过7天的培养后, 我们在 RCP 载体上获得了大量可行的细胞, 由活/死染色确定。这些结果通过 SEM 分析证实, 在毛孔周围观察完全地被殖民的载体, 部分 overgrowing 他们 (图 1B)。另一方面, 实验中的细胞不均匀播种导致了几个空载体。失败的实验的特征是异常高的死细胞数量不应超过总细胞数量的 10% (…
Discussion
载体的一个主要目标是细胞的扩展, 同时保持它们的分化, 以便将细胞送到需要的地方。所代表的方法引入 RCP 载体, 细胞能够附着, 增殖, 并殖民载体高细胞密度。这是观察活/死染色, 其中超过90% 的存活细胞被检测, 而只有少数死细胞获得后, 7 天的动态文化。同样, SEM 图像证实, 细胞覆盖整个表面的载体后, 7 天的文化。
为了确保3D 模型中的细胞存活, 重要的是要保持细胞的氧?…
Declarações
The authors have nothing to disclose.
Acknowledgements
导致这些结果的研究得到了欧洲联盟第七框架方案 FP7/2007-2013 根据赠款协议编号 607051 (生物启发) 的资助。我们感谢 Carolien van Spreuwel-Goossens 从富士制造欧洲 bv 公司, 为技术援助在 RCP 制造, 和沃纳 Stracke 从弗劳恩霍夫研究所的硅酸盐研究 ISC, 以协助与 SEM 分析。
Materials
| 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) | Serva Electrophoresis GmbH | 20395.01 | |
| 4’,6-Diamidino-2-phenylindoldihydrochloride (DAPI) | Sigma-Aldrich | D9542 | |
| Acetic acid 100% | Sigma-Aldrich | 533,001 | |
| Analytical balance Kern EG 2200-2NM | Kern & Sohn GmbH | ||
| Ascorbate-2-phosphate | Sigma-Aldrich | A8960 | |
| Bioreactor | Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany | ||
| Bright field microscope Axiovert 40C | Carl Zeiss AG | ||
| Cellnest | Fujifilm | ||
| Centrifuge tubes (15 mL, 50 mL) | Greiner Bio-One | ||
| Collagen R solution 0,4% | Serva Electrophoresis GmbH | 47254.01 | |
| DMEM-F12 | Gibco | 11320-033 | |
| Dulbecco's Phosphate Buffered Saline | Sigma-Aldrich | D8537 | Modified, without calcium chloride and magnesium chloride |
| Eosin 1% | Morphisto | 10177.01000 | |
| Ethanol 96% | Carl Roth GmbH | T171.4 | Denatured |
| Fetal calf serum (FCS) | Bio&SELL | FCS.ADD.0500 | not heat-inactivated |
| Fluorescence microscope BZ-9000 | Keyence | ||
| Haematoxylin | Morphisto | 10231.01000 | |
| Hexamethyldisilazane | Sigma-Aldrich | 440191 | Reagent grade, ≥99% |
| Incubator for bioreactor | Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany | ||
| Live/Dead Cell Double Staining Kit | Fluka | 04511KT-F | |
| Magnetic stirrer plate | 2Mag | 80002 | |
| Medium 199 | Sigma-Aldrich | M0650 | 10X |
| Microplate reader Tecan Infinite M200 |
Tecan | ||
| Needle 21G 16mm | VWR | 613-5389 | |
| Papain from papaya latex | Sigma-Aldrich | P4762 | lyophilized powder, ≥ 10 units/mg protein |
| Paraffin | Carl Roth GmbH | 6642.6 | |
| Penicillin/Streptomycin | Sigma-Aldrich | P4333 | |
| Peristaltic pump | Ismatec | ||
| Quanti-iT PicoGreen dsDNA assay kit | Thermo Fischer Scientific | P7589 | |
| Histofix 4% | Carl Roth GmbH | P087 | |
| Scanning Electron Microscope Supra 25 | Carl Zeiss AG | ||
| Sodium hydroxide solution 1.0 N | Sigma-Aldrich | S2770 | |
| Spinner flasks (25 mL) | Wheaton | 356879 | |
| Syringe 1 mL | VWR | 720-2561 | |
| Tissue culture flasks (25 cm2, 75 cm2, 150 cm2) | TPP Techno Plastik Products AG | ||
| Trypan blue 0.4% | Sigma-Aldrich | T8154 | |
| VascuLife VEGF-Mv | Lifeline cell technology | LL-0005 |
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Citar este artigo
Suarez Muñoz, M., Confalonieri, D., Walles, H., van Dongen, E. M. W. M., Dandekar, G. Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model. J. Vis. Exp. (132), e57363, doi:10.3791/57363 (2018).