JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Résultats
Discussion
Divulgations
Acknowledgements
Materials
References
Traduction automatique
Please note that all translations are automatically generated. Click here for the English version.
Bio-ingénierie

牙齿和呼吸细胞及器官在微重力下的繁殖

Published: May 25, 2021
doi:
10.3791/62690
, , , ,
1Center for Craniofacial Research and Diagnosis,Texas A&M College of Dentistry, 2Department of Stomatology,The Fourth Affiliated Hospital, Harbin Medical University, 3Key Laboratory of Oral Medicine,Guangzhou Insititute of Oral Disease, Stomatology Hospital of Guangzhou Medical University

Summary

该协议提出了一种在微重力下培养和3D生长釉质母细胞样细胞的方法，以保持其细长和极化形状以及牙釉质特异性蛋白质表达。还描述了微重力下牙周工程结构和肺器官培养的培养条件。

Abstract

重力是人体细胞功能、增殖、细胞骨架结构和取向的关键决定因素之一。旋转生物反应器系统（RCCS）模拟在太空中发生的重力损失，而是通过连续旋转培养的细胞或组织来提供微重力环境。这些RCCS确保营养，生长和转录因子以及氧气的不间断供应，并解决了静止的2D（二维）细胞或器官培养皿中重力的一些缺点。在本研究中，我们使用RCCS共培养宫颈环细胞和牙髓细胞成为釉质母细胞，表征牙周祖细胞/支架相互作用，并确定炎症对肺泡的影响。RCCS环境促进了釉母细胞样细胞的生长，促进了牙周祖细胞增殖以响应支架涂层，并允许评估炎症变化对培养的肺泡的影响。这份手稿总结了沿途的环境条件、材料和步骤，并突出了关键方面和实验细节。总之，RCCS是掌握 体外 细胞培养和3D（三维）生长的创新工具，并允许研究不适合经典2D培养环境的细胞系统或相互作用。

Introduction

重力影响地球上生命的方方面面，包括单个细胞的生物学及其在生物体内的功能。细胞通过机械感受器感知重力，并通过重新配置细胞骨架结构和改变细胞分裂来响应重力的变化1，2，3。微重力的其他影响包括流体填充囊泡中的静水压力，细胞器的沉降以及浮力驱动的流动和热量对流4。关于重力损失对人体细胞和器官影响的研究最初是为了模拟太空飞行任务期间宇航员的失重环境5。然而，近年来，这些最初由NASA开发的用于模拟微重力的3D生物反应器技术正变得越来越重要，作为细胞群培养的新方法，否则不适合2D培养系统。

3D生物反应器通过在悬浮液中培养细胞来模拟微重力，从而产生持续的“自由落体”效果。旋转生物反应器的其他优点包括器官培养系统中没有空气暴露，剪切应力和湍流减少，以及持续暴露于不断变化的营养供应中。旋转细胞培养系统（RCCS）生物反应器提供的这些动态条件有利于空间共定位和单细胞聚集体的三维组装6，7。

以前的研究已经证明了旋转生物反应器在骨再生8，牙胚培养9和人类牙囊细胞培养10方面的优势。还有一份报告表明，RCCS增强EOE细胞增殖和分化为釉母细胞11。然而，分化的细胞被认为是基于釉质母细胞蛋白免疫荧光和/或仅釉质生成素表达的釉质母细胞11 ，而不考虑其细长的形态或极化细胞形状。

除了NASA开发的旋转壁容器（RWV）生物反应器外，从细胞生成3D聚集体的其他技术包括磁悬浮，随机定位机（RPM）和clinostat12。为了实现磁悬浮，用磁性纳米颗粒标记的细胞使用外部磁力悬浮，从而形成无支架的3D结构，这些结构已用于脂肪细胞结构的生物制造13，14，15。模拟微重力的另一种方法是通过控制围绕两个轴的同时旋转来产生多向 G 力，从而消除称为 clinostat16 的设备中心的累积重力矢量。当骨髓干细胞在抑溪恒温器中培养时，通过抑制成骨细胞分化来抑制新的骨形成，说明了微重力16的去分化作用之一。

促进釉质母细胞忠实培养的体外系统将为牙釉质组织工程迈出重要一步17。不幸的是，到目前为止，鳞翅目鳞翅目到目前为止，已经报道了五种不同的釉质母细胞样细胞系，包括小鼠釉质母细胞系细胞系（ALC），大鼠牙齿上皮细胞系（HAT-7），小鼠LS8细胞系20，猪PABSo-E细胞系21和大鼠SF2-24细胞系22。然而，这些细胞中的大多数在2D培养中失去了其独特的极化细胞形状。

在本研究中，我们转向旋转细胞培养生物反应器系统（RCCS），以促进与间充质祖细胞共培养的宫颈环上皮的釉母细胞样细胞的生长，并克服2D培养系统的挑战，包括营养物质流动减少和重力引起的细胞骨架变化。此外，RCCS为研究与牙周组织工程相关的细胞/支架相互作用以及检查炎症介质对体 外肺泡组织的影响提供了新的途径。总之，这些研究的结果强调了基于微重力的旋转培养系统在分化上皮的繁殖和评估环境对 体外生长细胞的影响方面的益处，包括细胞/支架相互作用和组织对炎症状况的反应。

Protocol

获得了所有必要的机构批准，以确保该研究符合TAMU机构动物护理指南。 1. 生物反应器组装和灭菌 按照制造商的建议，在塑料仪器循环的高压釜中对生物反应器的四个高纵横比容器（HARV）进行灭菌20分钟。 灭菌后，通过拧紧生物反应器随附的螺钉将容器组装在细胞培养罩中，然后容器即可使用。 2. 用于生物反应器共培养实验的支架 <…

Representative Results

生物反应器的内室为细胞增殖和分化、附着在支架上或聚集形成类似组件的组织提供了环境。每个 HARV 容器可容纳多达 10 mL 的培养基，并促进营养物质的持续循环，使每个细胞都有极好的存活机会。图1A说明了注射器端口与连接无菌一站式阀的容器前板上的连接。这些阀门充当培养室的守门人。介质更换需要打开截止旋塞阀，以方便连接含有新鲜培养基的无菌注射器。生物?…

Discussion

在微重力下细胞生长方案的关键步骤包括生物反应器，支架，用于3D培养的细胞以及作为诱导细胞分化手段的支架涂层。我们研究中使用的生物反应器类型包括RCCS-4生物反应器，这是NASA开发的原始旋转细胞培养系统（RCCS）旋转圆柱形组织培养装置的最新修改，用于在模拟微重力下生长细胞。这种RCCS-4环境提供了极低的剪切应力，从而增强了传质并改善了培养性能。这个版本的生物反应器允许同时…

Divulgations

The authors have nothing to disclose.

Acknowledgements

研究得到了国家牙科和颅面研究所（UG3-DE028869和R01-DE027930）的慷慨资助。

Materials

Antibiotic-antimycotic ThermoFisher Scientfic 15240096
Ascorbic Acid Sigma Aldrich A4544
BGJb Fitton-Jackson Modification media ThermoFisher Scientfic 12591
BIOST PGA scaffold Synthecon Custom Available from the company through a custom order
BMP-2 R&D Systems 355-BM
BMP-4 R&D Systems 314-BP
DMEM Media Sigma Aldrich D6429-500mL
FBS ThermoFisher Scientfic 16140071
Fibricol Advanced Biomatrix 5133-20mL
Fibronectin Corning 354008
Galanin Sigma Aldrich G-0278
Gelatin disc Advanced Biomatrix CytoForm 500
Graphene sheets Advanced Biomatrix CytoForm 300
hEGF Peprotech AF-100-15
hFGF ThermoFisher Scientfic AA1-155
Hydroxyapatite disc Advanced Biomatrix CytoForm 200
Il-6 protein PeproTech 200-06
Keratinocyte SFM media (1X) ThermoFisher Scientfic 17005042
Laminin Corning 354259
LRAP peptide Peptide 2.0 Custom made sequence: MPLPPHPGSPGYINLSYEVLT
PLKWYQSMIRQPPLSPILPEL
PLEAWPATDKTKREEVD
Matrigel Corning 354234
Millipore Nitrocellulose membrane Merck Millipore AABP04700
RCCS Bioreactor Synthecon RCCS 4HD
SpongeCol Advanced Biomatrix 5135-25EA
Syring valve one way stopcock w/swivel male luer lock Smiths Medical MX5-61L
Syringes with needle 3cc McKESSON 16-SN3C211
Trypsin EDTA (0.25%) ThermoFisher Scientfic 25200056
DOWNLOAD MATERIALS LIST

References

  1. Horneck, G., et al. Life sciences: microorganisms in the space environment. Science. 225 (4658), 226-228 (1984).
  2. Helmstetter, C. E. Gravity and the orientation of cell division. Proceedings of the National Academy of Sciences. 94 (19), 10195-10198 (1997).
  3. Bizzarri, M., Monici, M., van Loon, J. J. W. A. How microgravity affects the biology of living systems. BioMed Research International. 2015, 863075 (2015).
  4. Freed, L. E., Vunjak-Novakovic, G. Spaceflight bioreactor studies of cells and tissues. Advances in Space Biology and Medicine. 8, 177-195 (2002).
  5. Walther, I. Space bioreactors and their applications. Advances in Space Biology and Medicine. 8, 197-213 (2002).
  6. Morabito, C., et al. RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures. BioMed Research International. 2015, 754283 (2015).
  7. Schwarz, R. P., Goodwin, T. J., Wolf, D. A. Cell culture for three-dimensional modeling in rotating-wall vessels: An application of simulated microgravity. Journal of Tissue Culture Methods. 14 (2), 51-57 (1992).
  8. Nokhbatolfoghahaei, H., et al. Computational modeling of media flow through perfusion-based bioreactors for bone tissue engineering. Proceedings of the Institution of Mechanical Engineers, Part H. 234 (12), 1397-1408 (2020).
  9. Sun, F. -. y., Wang, X. -. m., Li, X. -. y. An innovative membrane bioreactor (MBR) system for simultaneous nitrogen and phosphorus removal. Process Biochemistry. 48 (11), 1749-1756 (2013).
  10. Steimberg, N., et al. Advanced 3D Models Cultured to Investigate Mesenchymal Stromal Cells of the Human Dental Follicle. Tissue Engineering Methods (Part C). 24 (3), 187-196 (2018).
  11. Li, P., et al. RCCS enhances EOE cell proliferation and their differentiation into ameloblasts. Molecular Biology Reports. 39 (1), 309-317 (2012).
  12. Grimm, D., et al. Tissue engineering under microgravity conditions-use of stem cells and specialized cells. Stem Cells and Development. 27 (12), 787-804 (2018).
  13. Tasoglu, S., et al. Magnetic Levitational Assembly for Living Material Fabrication. Advanced Healthcare Materials. 4 (10), 1469-1476 (2015).
  14. Sarigil, O., et al. Scaffold-free biofabrication of adipocyte structures with magnetic levitation. Biotechnology and Bioengineering. 118 (3), 1127-1140 (2021).
  15. Anil-Inevi, M., et al. Biofabrication of in situ Self Assembled 3D Cell Cultures in a Weightlessness Environment Generated using Magnetic Levitation. Scientific Reports. 8 (1), 7239 (2018).
  16. Nishikawa, M., et al. The effect of simulated microgravity by three-dimensional clinostat on bone tissue engineering. Cell Transplant. 14 (10), 829-835 (2005).
  17. Pandya, M., Diekwisch, T. G. H. Enamel biomimetics-fiction or future of dentistry. International Journal of Oral Science. 11 (1), 8 (2019).
  18. Klein, O. D., et al. Meeting report: a hard look at the state of enamel research. International Journal of Oral Science. 9 (11), 3 (2017).
  19. Liu, H., Yan, X., Pandya, M., Luan, X., Diekwisch, T. G. Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium. Stem Cells and Development. 25 (20), 1580-1590 (2016).
  20. Chen, L. S., Couwenhoven, R. I., Hsu, D., Luo, W., Snead, M. L. Maintenance of amelogenin gene expression by transformed epithelial cells of mouse enamel organ. Archives of Oral Biology. 37 (10), 771-778 (1992).
  21. DenBesten, P. K., Gao, C., Li, W., Mathews, C. H., Gruenert, D. C. Development and characterization of an SV40 immortalized porcine ameloblast-like cell line. European Journal of Oral Sciences. 107 (4), 276-281 (1999).
  22. Arakaki, M., et al. Role of epithelial-stem cell interactions during dental cell differentiation. Journal of Biological Chemistry. 287 (13), 10590-10601 (2012).
  23. Au – Chavez, M. G., et al. Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor. Journal of Visualized Experiments. (87), e51266 (2014).
  24. Pandya, M., et al. Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development. Frontiers in Physiology. 8, 790 (2017).
  25. Dangaria, S. J., Ito, Y., Luan, X., Diekwisch, T. G. Differentiation of neural-crest-derived intermediate pluripotent progenitors into committed periodontal populations involves unique molecular signature changes, cohort shifts, and epigenetic modifications. Stem Cells and Development. 20 (1), 39-52 (2011).
  26. Ahadian, S., et al. Electrical stimulation as a biomimicry tool for regulating muscle cell behavior. Organogenesis. 9 (2), 87-92 (2013).
  27. Smith, C. E. Cellular and chemical events during enamel maturation. Critical Reviews in Oral Biology & Medicine. 9 (2), 128-161 (1998).
  28. Pei, M., et al. Bioreactors mediate the effectiveness of tissue engineering scaffolds. The FASEB Journal. 16 (12), 1691-1694 (2002).
  29. Ahmed, S., Chauhan, V. M., Ghaemmaghami, A. M., Aylott, J. W. New generation of bioreactors that advance extracellular matrix modelling and tissue engineering. Biotechnology Letters. 41 (1), 1-25 (2019).
  30. Seidel, K., et al. Resolving stem and progenitor cells in the adult mouse incisor through gene co-expression analysis. Elife. 6, (2017).
  31. Green, H., Rheinwald, J. G., Sun, T. T. Properties of an epithelial cell type in culture: the epidermal keratinocyte and its dependence on products of the fibroblast. Progress in Clinical and Biological Research. 17, 493-500 (1977).
  32. Green, H. The birth of therapy with cultured cells. Bioessays. 30 (9), 897-903 (2008).
  33. Zeichner-David, M., et al. Control of ameloblast differentiation. International Journal of Developmental Biology. 39 (1), 69-92 (1995).
  34. Bei, M., Stowell, S., Maas, R. Msx2 controls ameloblast terminal differentiation. Developmental Dynamics. 231 (4), 758-765 (2004).
  35. Dangaria, S. J., Ito, Y., Luan, X., Diekwisch, T. G. Successful periodontal ligament regeneration by periodontal progenitor preseeding on natural tooth root surfaces. Stem Cells and Development. 20 (10), 1659-1668 (2011).
  36. Del Moral, P. M., Warburton, D. Explant culture of mouse embryonic whole lung, isolated epithelium, or mesenchyme under chemically defined conditions as a system to evaluate the molecular mechanism of branching morphogenesis and cellular differentiation. Methods in Molecular Biology. 633, 71-79 (2010).
  37. Hermanns, M. I., Unger, R. E., Kehe, K., Peters, K., Kirkpatrick, C. J. Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro. Laboratory Investigation. 84 (6), 736-752 (2004).
  38. Duell, B. L., Cripps, A. W., Schembri, M. A., Ulett, G. C. Epithelial cell coculture models for studying infectious diseases: benefits and limitations. Journal of Biomedicine and Biotechnolog. 2011, 852419 (2011).
  39. Kuppan, P., Sethuraman, S., Krishnan, U. M. In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds. Materials Science and Engineering: C. 81, 191-205 (2017).
  40. Navran, S. The application of low shear modeled microgravity to 3-D cell biology and tissue engineering. Biotechnology Annual Review. 14, 275-296 (2008).

Tags

MicrogravityDental CellsRespiratory CellsAmeloblastEnamelBioreactorScaffoldMedia ChangeMouse MandibleCervical LoopDental PulpEnamel Research
check_url/fr/62690?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citer Cet Article
Pandya, M., Ma, W., Lyu, H., Luan, X., Diekwisch, T. G. H. Propagation of Dental and Respiratory Cells and Organs in Microgravity. J. Vis. Exp. (171), e62690, doi:10.3791/62690 (2021).
Télécharger la Citation
Réimpressions et Autorisations

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image