自动点胶机器人技术在细胞表面和指导生物印刷
Summary
This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.
Abstract
这个手稿描述引进细胞指导功能接着用自动机器人分配系统对这些特征的直接递送细胞在水凝胶生物油墨。被选定的特定生物油墨，因为它使细胞对沉积物和感知功能。分配系统bioprints使用背压辅助打印头中的水凝胶bioinks活细胞。然而，通过用锋利笔或手术刀更换打印头，分配系统也可以采用以创建经过表面刻蚀地形线索。触针运动可以在X，Y的10微米的步骤和Z方向进行编程。图案化的凹槽能够定向间质干细胞，影响它们通过在对准的细长的形态与凹槽'方向。图案形成可以在直线，同心圆，和正弦波使用绘图软件来设计。在随后的过程中，成纤维爆炸和间充质干细胞悬浮于2％的明胶生物油墨，用于在背压从动挤压印刷头生物印刷。然后将细胞轴承生物油墨使用用于蚀刻相同的编程坐标打印。所述bioprinted细胞能够感知和通过沿刻蚀槽的方向上其细长方向所证明反应的蚀刻特征。
Introduction
单元布局的蓄意构图，可允许在体内细胞组织1模仿文化的形成。事实上，研究多种细胞类型之间的相互作用可以通过组织他们的空间位置2,3的协助。大多数图形系统依赖于表面改性方法，为推动或阻止随后的被动细胞沉积的细胞粘附。提供生物印刷在细胞分布1空间和时间的控制。除了这些功能，生物印刷已被描述为用于产生几何形状复杂的支架4在技术上简单，快速和成本有效的方法。它利用计算机设计软件，并允许引入细胞进入制造工序4。
生物印刷系统已根据自己的基础工作原理激光被分类，喷墨型或挤压型4。挤压生物印刷已被描述为是最有前途的，因为它允许一个现实的时间帧4-6内临床相关尺寸的组织构建物的制造。它是由一个细胞轴承水凝胶生物油墨的机械或背压辅挤出进行。在这里提出的方法中，被采用的背压。如所提到的，将细胞在细胞相容性生物油墨递送。这样的生物油墨应该支持细胞的递送，而不产生有害的剪切应力，并具有足够的粘度以保持在印刷迹的完整性，而不会破裂或扩散（称为“墨渗色”），7-10。
已知细胞与它们的贴壁表面的相互作用来影响细胞的行为。表面形貌可以控制细胞形状，方向11，甚至表型。尤其是，沟槽和通道的制造中已被证明诱导在多个细胞类型拉伸的，细长的形态。这种形态的通过已发现影响多能和多潜能细胞的表型。例如，当在槽对齐，间质干细胞（MSC）表现出对心肌12,13和血管平滑肌细胞分化的证据采用收缩表型在合成10,14-17。
可在聚合物表面通过许多方法来产生对准通道或沟槽的细胞，例如，深反应离子蚀刻，电子束光刻，激光直接印刷，飞秒激光，光刻和等离子干蚀刻18。这些方法通常耗时，需要复杂的设备，并且可以在所产生的图案的形状进行限制。此外，他们不与同步生物印刷图案，不允许立即细胞化。一个自动化的协调控制的运动分配系统可以遵循复杂的图案为溶液的沉积。在这里，我们展示了微型受控的运动是如何被利用来创造细胞定向通道。削尖触笔或手术刀被连接到所述打印头代替挤出注射器中，然后将设备可以蚀刻绘图软件的指导下，在聚合物表面上。该方法在图案设计提供了多功能性，并适用于在生物工程常用如聚苯乙烯，聚四氟乙烯，和聚己内酯的聚合物材料。作为后续步骤的蚀刻，细胞可以直接bioprinted到划伤凹槽。这里使用的明胶生物油墨能够既保持跟踪，并允许沉积的细胞来检测蚀刻特征。 bioprinted的刻蚀槽间充质干细胞，表现在不同的线条沿它们伸长。
Protocol
Representative Results
Discussion
这个过程的关键步骤是干细胞作为过程必须允许细胞沉降到该特征，打印无生物油墨扩频/出血，递送细胞无剪切应力的细胞死亡，而不是触发分化朝不希望的谱系的实际生物印刷递送。
如果预期细胞对准未能发生，则该生物油墨粘度应该评估其适合印刷。重要的是，该生物油墨允许细胞沉降到该图案化的聚合物表面上。该生物油墨聚合物的浓度，可以减少或它的温度升?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).
Materials
| Equipment | |||
| Robotic Dispensing System | Janome | 2300N | |
| Plasma Machine | Femto Science | Covance | |
| USB Microscope | |||
| Optical Microscope | Olympus | IX71 | |
| Name | Company | Catalog Number | Comments |
| Software | |||
| Spreadsheet | Excel | Excel | |
| Printing Co-ordinate Software | Janome | JR C-Points | |
| Imaging Software | National Institutes of Health (NIH) | ImageJ | |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Stylus (Blade) | OLFA | AK-5 | |
| 5ml printing syringe | San-ei Tech | SH10LL-B | |
| 30G printing needle | San-ei Tech | SH30-0.25-B | |
| 1mm polystyrene sheets | Purchased locally | ||
| Fetal bovine serum | Invitrogen | 10270-098 | |
| Phosphate buffered saline | Invitrogen | ||
| Gelatin from porcine skin, Gel strength 300, Type A | Sigma Aldrich | 9000-70-8 | |
| αMEM | Invitrogen | 41061-029 | |
| Antibiotc antimycotic | Sigma Aldrich | A5955-100ML | |
| Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs) | Cyagen Biosciences Incorporation | RASMX-01201 |
Riferimenti
- Ma, Z., Ringeisen , R. B., Spargo, J. B., Wu, K. P., et al. . Cell and Organ Printing. , 137-159 (2010).
- Kaji, H., Camci-Unal, G., Langer, R., Khademhosseini, A. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions. Biochim Biophys Acta. 1810 (3), 239-250 (2011).
- Goubko, C. A., Cao, X. Patterning multiple cell types in co-cultures: A review. Materials Science and Engineering: C. 29 (6), 1855-1868 (2009).
- Pati, F., Jang, J., Lee, J. W., Cho, D. W., Yoo, J. J. . Essentials of 3D Biofabrication and Translation. , 123-152 (2015).
- Derby, B. Printing and prototyping of tissues and scaffolds. Science. 338 (6109), 921-926 (2012).
- Malda, J., et al. 25th anniversary article: Engineering hydrogels for biofabrication. Adv Mater. 25 (36), 5011-5028 (2013).
- Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials. 33 (26), 6020-6041 (2012).
- Skardal, A., Yoo, J. J. . Essentials of 3D Biofabrication and Translation. , 1-17 (2015).
- Irvine, S. A., et al. Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking. Biomed Microdevices. 17 (1), 16 (2015).
- Bhuthalingam, R., et al. A novel 3D printing method for cell alignment and differentiation. International Journal of Bioprinting. 1, 57-65 (2015).
- Curtis, A., Wilkinson, C. Topographical control of cells. Biomaterials. 18 (24), 1573-1583 (1997).
- Tay, C. Y., et al. Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage. Exp Cell Res. 316 (7), 1159-1168 (2010).
- Tay, C. Y., et al. Bio-inspired micropatterned platform to steer stem cell differentiation. Small. 7 (10), 1416-1421 (2011).
- Li, H., et al. Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells. Acta Biomater. 8 (2), 531-539 (2012).
- Kolind, K., Leong, K. W., Besenbacher, F., Foss, M. Guidance of stem cell fate on 2D patterned surfaces. Biomaterials. 33 (28), 6626-6633 (2012).
- Agrawal, A., et al. Smooth Muscle Cell Alignment and Phenotype Control by Melt Spun Polycaprolactone Fibers for Seeding of Tissue Engineered Blood Vessels. International Journal of Biomaterials. 2015, (2015).
- Chang, S., et al. Phenotypic modulation of primary vascular smooth muscle cells by short-term culture on micropatterned substrate. PLoS One. 9 (2), e88089 (2014).
- Li, Y., et al. Engineering cell alignment in vitro. Biotechnol Adv. 32 (2), 347-365 (2014).
- Blaeser, A., et al. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity. Adv Healthc Mater. 5 (3), 326-333 (2016).
- Nery, A. A., et al. Human mesenchymal stem cells: From immunophenotyping by flow cytometry to clinical applications. Cytometry Part A. 83A (1), 48-61 (2013).
- Guvendiren, M., Lu, H. D., Burdick, J. A. Shear-thinning hydrogels for biomedical applications. Soft Matter. 8 (2), 260-272 (2012).
- Markstedt, K., et al. 3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications. Biomacromolecules. 16 (5), 1489-1496 (2015).
- Kolesky, D. B., et al. 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater. 26 (19), 3124-3130 (2014).
- Merceron, T. K., Murphy, S. V., Yoo, J. J. . Essentials of 3D Biofabrication and Translation. , 249-270 (2015).
- Carrow, J. K., Kerativitayanan, P., Jaiswal, M. K., Lokhande, G., Gaharwar, A. K., Yoo, J. J. . Essentials of 3D Biofabrication and Translation. , 229-248 (2015).
- Lee, K. V., Turksen, K., et al. . Bioprinting in Regenerative Medicine. , 33-66 (2015).
- Ozbolat, I. T., Hospodiuk, M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials. 76, 321-343 (2016).
- Li, M., Tian, X., Schreyer, D. J., Chen, X. Effect of needle geometry on flow rate and cell damage in the dispensing-based biofabrication process. Biotechnol Prog. 27 (6), 1777-1784 (2011).
- Nikkhah, M., Edalat, F., Manoucheri, S., Khademhosseini, A. Engineering microscale topographies to control the cell-substrate interface. Biomaterials. 33 (21), 5230-5246 (2012).
- Khademhosseini, A., et al. Microfluidic patterning for fabrication of contractile cardiac organoids. Biomed Microdevices. 9 (2), 149-157 (2007).
- Chelli, B., et al. Neural cell alignment by patterning gradients of the extracellular matrix protein laminin. Interface Focus. 4 (1), 20130041 (2014).
- Aubin, H., et al. Directed 3D cell alignment and elongation in microengineered hydrogels. Biomaterials. 31 (27), 6941-6951 (2010).
- Béduer, A., et al. Engineering of adult human neural stem cells differentiation through surface micropatterning. Biomaterials. 33 (2), 504-514 (2012).
- Bian, W., Jackman, C. P., Bursac, N. Controlling the structural and functional anisotropy of engineered cardiac tissues. Biofabrication. 6 (2), 024109 (2014).
- Chen, P. Y., et al. Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase. Acta Biomater. 10 (2), 912-920 (2014).
- Bertassoni, L. E., et al. Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels. Biofabrication. 6 (2), 024105 (2014).
- Zhao, X., et al. 3D patterned substrates for bioartificial blood vessels – The effect of hydrogels on aligned cells on a biomaterial surface. Acta Biomater. 26, 159-168 (2015).
