JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Bioengineering

Mikrofluid Bioprinting for Engineering vaskulariserede væv og Organoids

Published: August 11, 2017
doi:
10.3791/55957
, ,
1Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,Harvard Medical School, 2Department of Plastic and Reconstructive Surgery, Renji Hospital,Shanghai Jiao Tong University School of Medicine, 3Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences,Utrecht University

Summary

Vi leverer en generaliseret protokol baseret på en mikrofluid bioprinting strategi for engineering en microfibrous vaskulære seng, hvor en secondary celletype kunne blive yderligere seedede i det interstitielle rum af denne microfibrous struktur til at generere vaskulariserede væv og organoids.

Abstract

Engineering vaskulariserede væv konstruktioner og organoids har været historisk udfordrende. Her beskriver vi en roman metode baseret på mikrofluid bioprinting til at generere et stillads med flerlaget interlacing hydrogel mikrofibre. For at opnå glat var bioprinting, en kerne-kappe mikrofluid printhovedet med en composite bioink formulering ekstruderet fra core flow og den crosslinking løsning udført af kappe flow, designet og monteres på bioprinter. Ved at blande gelatine methacryloyl (GelMA) med natriumalginat, en polysaccharid, der undergår øjeblikkelig ionisk crosslinking i Vælg divalent ioner, efterfulgt af en sekundær photocrosslinking i komponenten GelMA at opnå permanent stabilisering, en microfibrous stillads kunne være opnået ved hjælp af denne bioprinting strategi. Vigtigere, kan endotelceller indkapslet inde bioprinted mikrofibre danne lumen-lignende strukturer, der ligner Vaskulaturen i løbet af kultur i 16 dage. Endothelialized microfibrous stillads kan bruges yderligere som en vaskulær seng til at konstruere en vaskulariserede væv gennem efterfølgende såning af typen secondary celle i det interstitielle rum af af mikrofibre. Mikrofluid bioprinting giver en generel strategi i praktisk engineering af vaskulariserede væv på high fidelity.

Introduction

Tissue engineering mål at skabe funktionelle væv erstatninger, der kan bruges til at udskifte, genoprette eller forøge de tilskadekomne eller syge i den menneskelige krop1,2,3,4, ofte gennem en kombination af ønskede celletyper, bioaktive molekyler5,6, og biomaterialer7,8,9,10. Mere nylig, tissue engineering teknologier er også i stigende grad vedtaget for at generere in vitro- væv og orgel modeller, der efterligner de vigtige funktioner i deres i vivo modparter, til applikationer såsom udvikling af lægemidler, i stedet for de konventionelle forenklede planar celle kulturer11,12,13,14,15,16,17,18,19. I begge situationer, evne til at sammenfatte de komplekse mikroarkitektur og hierarkisk struktur af de humane væv er kritisk i at aktivere funktionaliteten af manipuleret væv10, og navnlig, måder at integrere en vaskulære netværk i manipuleret væv efterspørgslen siden vascularization præsenterer en af de største udfordringer til felt20,21,22,23.

Til dato, en række forskellige metoder er blevet udviklet i denne henseende i et forsøg på at opbygge strukturer, blodkar i manipuleret væv konstruktioner med forskellige grader af succes8. For eksempel, samlesæt i endothelial celler giver mulighed for generation af mikrovaskulære netværk24; levering af angiogene vækstfaktorer inducerer vedvarende neovascularization25,26; Brug af vaskulære stamceller og pericytes letter endotel cellevækst og forsamling24,27; designe stillads egenskaber giver præcise graduering af vascularization28,29; og celle ark teknologi giver mulighed for bekvem manipulation af vaskulære lagdeling30. Disse strategier dog ikke begave evne til at kontrollere den rumlige mønstre af Vaskulaturen, ofte fører til tilfældige fordeling af blod fartøjer inden for en manipuleret væv konstruere og dermed begrænsede reproducerbarhed. I løbet af de sidste par år opstået bioprinting som en klasse af teknologier til løsning af sådan en udfordring på grund af deres enestående alsidighed af deponeringen komplekse væv mønstre på high fidelity og reproducerbarhed i en automatiseret eller semi-automatiseret måde31,32,33. Hellige bioprinting34,35,36,37,38, integrerede bioprinting39,40,41, og hule struktur bioprinting/biofabrication42,43,44,45,46,47,48,49,50,51,52,53 har alle udvist gennemførligheden af generering af vaskulær eller vaskulariserede væv.

Alternativt, en mikrofluid bioprinting strategi at fabrikere microfibrous stilladser har udviklet for nylig, hvor en hybrid bioink sammensat af natriumalginat og gelatine methacryloyl (GelMA) blev leveret gennem kernen i en koncentrisk printhoved og en calciumchlorid (CaCl2) løsning blev gennemført den ydre kappe flow af printhovedet54,55. Co ekstrudering af de to strømme tilladt for umiddelbare fysiske crosslinking af komponenten calciumalginat til aktiverer microfiber dannelse, mens efterfølgende photocrosslinking sikres permanent stabilisering af flere lag microfibrous stillads. Af note fandtes endotelceller indkapslet i bioprinted mikrofibre til at formere sig og vandrer mod periferier af mikrofibre antager lumen-lignende strukturer, der efterlignede de vaskulære bed54,55. Disse bioprinted, endothelialized vaskulære senge kunne være senere befolket med ønskede sekundære celletyper til yderligere konstruere vaskulariserede væv55. Denne protokol giver således en detaljeret procedure for sådan en mikrofluid bioprinting strategi aktiveret tilsigtet koncentriske dyse, som sikrer bekvem fabrikation af vaskulariserede væv for potentielle anvendelser i både vævsmanipulering og organoid modellering.

Protocol

Neonatal rotte cardiomyocytes anvendes i denne protokol blev isoleret fra 2 dage gamle Sprague-Dawley rotter efter en veletableret procedure56 godkendt af institutionelle Animal Care og brug Udvalget på Brigham and Women’s Hospital. 1. instrumentering af Bioprinter Indsætte en mindre stumpt nål (f.eks., 27 G, 1 tomme) som kernen i centrum af en større stump nål (f.eks., 18 G, ½ tomme) som kappe at konstruere dual-layer, koncentriske m…

Representative Results

Mikrofluid bioprinting strategi giver mulighed for direkte ekstrudering bioprinting af microfibrous stilladser med lav viskositet bioinks54,55. Som illustreret i figur 2A, et stillads med en størrelse på 6 × 6 × 6 mm3 indeholdende > 30 lag af mikrofibre kunne være bioprinted i 10 min. Den umiddelbare ionisk crosslinking af komponenten calciumalginat med CaCl2<…

Discussion

Opførelsen af co-axial printhovedet er et kritisk skridt mod vellykket mikrofluid bioprinting at tillade samtidige levering af både bioink fra kernen og crosslinking agenten fra kappe. Mens i denne protokol et eksempel printhovedet blev oprettet ved hjælp af en 27G nål som kernen og en 18G nåle som shell, kan den let udvides til en række forskellige kombinationer med forskellige størrelser af nåle. Men ændringen i nål størrelser, hvilket resulterer i ændring i mængden af strøm leveret i hver fase, vil kræv…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne anerkende National Cancer Institute af den nationale institutter sundhed vej til uafhængighed Award (K99CA201603).

Materials

Alginic acid sodium salt from brown algae Sigma-Aldrich A0682 BioReagent, plant cell culture tested, low viscosity, powder
Gelatin type A from porcine skin Sigma-Aldrich G2500 Gel strength 300
Irgacure 2959 (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) Sigma-Aldrich 410896 98%
HEPES buffer Sigma-Aldrich H0887 1 M, pH 7.0-7.6, sterile-filtered, BioReagent, suitable for cell culture
Fetal bovine serum  Thermo Fisher Scientific 10438026 Qualified, heat-inactivated, USDA-approved regions
Calcium chloride dihydrate Sigma-Aldrich C5080 BioXtra, ≥99.0%
Phosphate buffered saline Thermo Fisher Scientific 10010023 pH 7.4
Human umbilical vein endothelial cells Angio-Proteomie cAP-0001 Human Umbilical Vein Endothelial Cells (HUVECs)
GFP-expressing human umbilical vein endothelial cells Angio-Proteomie cAP-0001GFP GFP-Expressing Human Umbilical Vein Endothelial Cells (GFPHUVECs)
Endothelial cell growth medium Lonza CC-3162 EGM-2 BulletKit
Dulbecco’s Modified Eagle Medium  Thermo Fisher Scientific 12430054 High glucose, HEPES
Sylgard 184 silicone elastomer kit Ellsworth Adhesives 184 SIL ELAST KIT 0.5KG Clear 0.5 kg Kit
UV curing lamp system Excelitas Technologies OmniCure S2000 Spot UV Light Curing System with Intelligent UV Sensor
DOWNLOAD MATERIALS LIST

References

  1. Langer, R., Vacanti, J. P. Tissue Engineering. Science. 260 (5110), 920-926 (1993).
  2. Khademhosseini, A., Vacanti, J. P., Langer, R. Progress in Tissue Engineering. Sci. Am. 300 (5), 64-71 (2009).
  3. Langer, R. Tissue Engineering: Status and Challenges. E-Biomed: J.Regen. Med. 1 (1), 5-6 (2004).
  4. Atala, A., Kasper, F. K., Mikos, A. G. Engineering Complex Tissues. Sci. Transl. Med. 4 (160), 112 (2012).
  5. Biondi, M., Ungaro, F., Quaglia, F., Netti, P. A. Controlled Drug Delivery in Tissue Engineering. Adv. Drug Del. Rev. 60 (2), 229-242 (2008).
  6. Tayalia, P., Mooney, D. J. Controlled Growth Factor Delivery for Tissue Engineering. Adv. Mater. 21 (32-33), 3269-3285 (2009).
  7. Hubbell, J. A. Biomaterials in Tissue Engineering. Nat. Biotechnol. 13 (6), 565-576 (1995).
  8. Place, E. S., Evans, N. D., Stevens, M. M. Complexity in Biomaterials for Tissue Engineering. Nat. Mater. 8 (6), 457-470 (2009).
  9. Rice, J. J., et al. Engineering the Regenerative Microenvironment with Biomaterials. Adv. Healthcare Mater. 2 (1), 57-71 (2012).
  10. Zhang, Y. S., Xia, Y. Multiple Facets for Extracellular Matrix Mimicking in Regenerative Medicine. Nanomedicine. 10 (5), 689-692 (2015).
  11. Huh, D., Hamilton, G. A., Ingber, D. E. From 3D Cell Culture to Organs-on-Chips. Trends Cell Biol. 21 (12), 745-754 (2011).
  12. Bhatia, S. N., Ingber, D. E. Microfluidic Organs-on-Chips. Nat. Biotechnol. 32 (8), 760-772 (2014).
  13. Esch, E. W., Bahinski, A., Huh, D. Organs-on-Chips at the Frontiers of Drug Discovery. Nat. Rev. Drug Discov. 14 (4), 248-260 (2015).
  14. Zhang, Y. S., Khademhosseini, A. Seeking the Right Context for Evaluating Nanomedicine: From Tissue Models in Petri Dishes to Microfluidic Organs-on-a-Chip. Nanomedicine. 10, 685-688 (2015).
  15. Zhang, C., Zhao, Z., Abdul Rahim, N. A., Van Noort, D., Yu, H. Towards a Human-on-Chip: Culturing Multiple Cell Types on a Chip with Compartmentalized Microenvironments. Lab Chip. 9 (22), 3185-3192 (2009).
  16. Moraes, C., Mehta, G., Lesher-Perez, S. C., Takayama, S. Organs-on-a-Chip: A Focus on Compartmentalized Microdevices. Ann. Biomed. Eng. 40 (6), 1211-1227 (2012).
  17. Sung, J. H., et al. Microfabricated Mammalian Organ Systems and Their Integration into Models of Whole Animals and Humans. Lab Chip. 13 (7), 1201-1212 (2013).
  18. Wikswo, J. P. The Relevance and Potential Roles of Microphysiological Systems in Biology and Medicine. Exp. Biol. Med. 239 (9), 1061-1072 (2014).
  19. Yum, K., Hong, S. G., Healy, K. E., Lee, L. P. Physiologically Relevant Organs on Chips. Biotechnol. J. 9 (1), 16-27 (2014).
  20. Nomi, M., Atala, A., Coppi, P. D., Soker, S. Principals of Neovascularization for Tissue Engineering. Mol. Aspects Med. 23 (6), 463-483 (2002).
  21. Jain, R. K., Au, P., Tam, J., Duda, D. G., Fukumura, D. Engineering Vascularized Tissue. Nat. Biotechnol. 23 (7), 821-823 (2005).
  22. Rouwkema, J., Rivron, N. C., Van Blitterswijk, C. A. Vascularization in Tissue Engineering. Trends Biotechnol. 26 (8), 434-441 (2008).
  23. Bae, H., et al. Building Vascular Networks. Sci. Transl. Med. 4 (160), 123 (2012).
  24. Rouwkema, J., Khademhosseini, A. Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks. Trends Biotechnol. 34 (9), 733-745 (2016).
  25. Perets, A., et al. Enhancing the Vascularization of Three-Dimensional Porous Alginate Scaffolds by Incorporating Controlled Release Basic Fibroblast Growth Factor Microspheres. J. Biomed. Mater. Res. A. 65 (4), 489-497 (2003).
  26. Davies, N. H., Schmidt, C., Bezuidenhout, D., Zilla, P. Sustaining Neovascularization of a Scaffold through Staged Release of Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-BB. Tissue Eng. A. 18 (1-2), 26-34 (2012).
  27. Sorrell, J. M., Baber, M. A., Caplan, A. I. Influence of Adult Mesenchymal Stem Cells on in Vitro Vascular Formation. Tissue Eng. A. 15 (7), 1751-1761 (2009).
  28. Quint, C., et al. Decellularized Tissue-Engineered Blood Vessel as an Arterial Conduit. Proct. Natl. Acad. Sci. U.S.A. 108 (22), 9214-9219 (2011).
  29. Choi, S. -. W., Zhang, Y., Macewan, M. R., Xia, Y. Neovascularization in Biodegradable Inverse Opal Scaffolds with Uniform and Precisely Controlled Pore Sizes. Adv. Healthcare Mater. 2 (1), 145-154 (2013).
  30. Sakaguchi, K., Shimizu, T., Okano, T. Construction of Three-Dimensional Vascularized Cardiac Tissue with Cell Sheet Engineering. J. Controlled Release. 205, 83-88 (2015).
  31. Zhang, Y. S., et al. 3D Bioprinting for Tissue and Organ Fabrication. Ann. Biomed. Eng. 45 (1), 148-163 (2017).
  32. Malda, J., et al. 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Adv. Mater. 25 (36), 5011-5028 (2013).
  33. Murphy, S. V., Atala, A. 3d Bioprinting of Tissues and Organs. Nat. Biotechnol. 32 (8), 773-785 (2014).
  34. Miller, J. S., et al. Rapid Casting of Patterned Vascular Networks for Perfusable Engineered Three-Dimensional Tissues. Nat. Mater. 11 (9), 768-774 (2012).
  35. Bertassoni, L. E., et al. Hydrogel Bioprinted Microchannel Networks for Vascularization of Tissue Engineering Constructs. Lab Chip. 14 (13), 2202-2211 (2014).
  36. Kolesky, D. B., et al. 3d Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs. Adv. Mater. 26 (19), 3124-3130 (2014).
  37. Lee, V. K., et al. Creating Perfused Functional Vascular Channels Using 3d Bio-Printing Technology. Biomaterials. 35 (28), 8092-8102 (2014).
  38. Zhang, Y. S., et al. Bioprinted Thrombosis-on-a-Chip. Lab Chip. 16, 4097-4105 (2016).
  39. Bhattacharjee, T., et al. Writing in the Granular Gel Medium. Science Advances. 1 (8), 1500655 (2015).
  40. Highley, C. B., Rodell, C. B., Burdick, J. A. Direct 3d Printing of Shear-Thinning Hydrogels into Self-Healing Hydrogels. Adv. Mater. 27 (34), 5075-5079 (2015).
  41. Hinton, T. J., et al. Three-Dimensional Printing of Complex Biological Structures by Freeform Reversible Embedding of Suspended Hydrogels. Science Advances. 1 (9), 1500758 (2015).
  42. Jia, W., et al. Direct 3d Bioprinting of Perfusable Vascular Constructs Using a Blend Bioink. Biomaterials. 106, 58-68 (2016).
  43. Zhang, Y., et al. In Vitro Study of Directly Bioprinted Perfusable Vasculature Conduits. Biomaterials Science. 3 (1), 134-143 (2015).
  44. Gao, Q., He, Y., Fu, J. -. Z., Liu, A., Ma, L. Coaxial Nozzle-Assisted 3D Bioprinting with Built-in Microchannels for Nutrients Delivery. Biomaterials. 61, 203-215 (2015).
  45. Cornock, R., Beirne, S., Thompson, B., Wallace, G. G. Coaxial Additive Manufacture of Biomaterial Composite Scaffolds for Tissue Engineering. Biofabrication. 6 (2), 025002 (2014).
  46. Duan, B., Hockaday, L. A., Kang, K. H., Butcher, J. T. 3D Bioprinting of Heterogeneous Aortic Valve Conduits with Alginate/Gelatin Hydrogels. J. Biomed. Mater. Res. A. 101 (5), 1255-1264 (2013).
  47. Skardal, A., et al. Photocrosslinkable Hyaluronan-Gelatin Hydrogels for Two-Step Bioprinting. Tissue Eng. A. 16 (8), 2675-2685 (2010).
  48. Li, S., et al. Direct Fabrication of a Hybrid Cell/Hydrogel Construct by a Double-Nozzle Assembling Technology. J. Bioact. Compatible Polym. 24 (3), 249-265 (2009).
  49. Visser, J., et al. Biofabrication of Multi-Material Anatomically Shaped Tissue Constructs. Biofabrication. 5 (3), 035007 (2013).
  50. Boyd, D. A., Adams, A. A., Daniele, M. A., Ligler, F. S. Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape. Journal of visualized experiments: JoVE. (83), e50958 (2014).
  51. Daniele, M. A., Adams, A. A., Naciri, J., North, S. H., Ligler, F. S. Interpenetrating Networks Based on Gelatin Methacrylamide and Peg Formed Using Concurrent Thiol Click Chemistries for Hydrogel Tissue Engineering Scaffolds. Biomaterials. 35 (6), 1845-1856 (2014).
  52. Daniele, M. A., Boyd, D. A., Adams, A. A., Ligler, F. S. Microfluidic Strategies for Design and Assembly of Microfibers and Nanofibers with Tissue Engineering and Regenerative Medicine Applications. Adv. Healthcare Mater. 4 (1), 11-28 (2015).
  53. Daniele, M. A., Radom, K., Ligler, F. S., Adams, A. A. Microfluidic Fabrication of Multiaxial Microvessels Via Hydrodynamic Shaping. RSC Advances. 4 (45), 23440-23446 (2014).
  54. Colosi, C., et al. Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low Viscosity Bioink. Adv. Mater. 28 (4), 677-684 (2015).
  55. Zhang, Y. S., et al. Bioprinting 3D Microfibrous Scaffolds for Engineering Endothelialized Myocardium and Heart-on-a-Chip. Biomaterials. 110, 45-59 (2016).
  56. Khademhosseini, A., et al. Microfluidic Patterning for Fabrication of Contractile Cardiac Organoids. Biomed. Microdevices. 9 (2), 149-157 (2007).
  57. Yue, K., et al. Synthesis, Properties, and Biomedical Applications of Gelatin Methacryloyl (GelMA) Hydrogels. Biomaterials. 73, 254-271 (2015).
  58. Loessner, D., et al. Functionalization, Preparation and Use of Cell-Laden Gelatin Methacryloyl-Based Hydrogels as Modular Tissue Culture Platforms. Nat. Protocols. 11 (4), 727-746 (2016).
  59. Aung, A., Theprungsirikul, J., Lim, H. L., Varghese, S. Chemotaxis-Driven Assembly of Endothelial Barrier in a Tumor-on-a-Chip Platform. Lab Chip. 16, 1886-1898 (2016).
  60. Shin, S. R., et al. A Bioactive Carbon Nanotube-Based Ink for Printing 2d and 3d Flexible Electronics. Adv. Mater. 28 (17), 3280-3289 (2016).
  61. Shin, S. R., et al. Aptamer-Based Microfluidic Electrochemical Biosensor for Monitoring Cell Secreted Cardiac Biomarkers. Anal. Chem. 88, 10019-10027 (2016).
  62. Zhang, Y. S., et al. Google Glass-Directed Monitoring and Control of Microfluidic Biosensors and Actuators. Sci. Rep. 6, 22237 (2016).
  63. Colosi, C., et al. Rapid Prototyping of Chitosan-Coated Alginate Scaffolds through the Use of a 3d Fiber Deposition Technique. J. Mater. Chem. B. 2 (39), 6779-6791 (2014).
  64. Zhu, W., et al. Direct 3D Bioprinting of Prevascularized Tissue Constructs with Complex Microarchitecture. Biomaterials. 124, 106-115 (2017).
  65. Yu, Y., Zhang, Y., Martin, J. A., Ozbolat, I. T. Evaluation of Cell Viability and Functionality in Vessel-Like Bioprintable Cell-Laden Tubular Channels. J. Biomech. Eng. 135 (9), 091011-091011 (2013).
  66. Zhang, Y., Yu, Y., Chen, H., Ozbolat, I. T. Characterization of Printable Cellular Micro-Fluidic Channels for Tissue Engineering. Biofabrication. 5 (2), 025004 (2013).
  67. Zhang, Y., Yu, Y., Ozbolat, I. T. Direct Bioprinting of Vessel-Like Tubular Microfluidic Channels. J. Nanotechnol. Eng. Med. 4 (2), 020902 (2013).
  68. Dolati, F., et al. In Vitro Evaluation of Carbon-Nanotube-Reinforced Bioprintable Vascular Conduits. Nanotechnology. 25 (14), 145101 (2014).
  69. Hansen, C. J., et al. High-Throughput Printing Via Microvascular Multinozzle Arrays. Adv. Mater. 25 (1), 96-102 (2013).

Tags

Microfluidic BioprintingOrganoidsVascular VatLiverSkinCancerBioprinterAlginateGelMAPhotoinitiatorHEPES BufferFBSCalcium ChlorideHUVECsDual Channel Syringe PumpCrosslinking

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Zhang, Y. S., Pi, Q., van Genderen, A. M. Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids. J. Vis. Exp. (126), e55957, doi:10.3791/55957 (2017).
Download Citation
Reprints and Permissions

View Video

✖

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

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image