미세 패턴 혈관 근육 박막의 확장 문화 미세 유체 Genipin 증착 기술
Summary
We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.
Abstract
The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.
Introduction
이러한 뇌 혈관 경련, 2, 3, 고혈압, 동맥 경화증 및 4와 같은 혈관 질환은 서서히 발전 전형적으로 사실상 만성적이며, 혈관 평활근 세포 (혈관 평활근 세포)로 역기능 힘 발생을 수반한다. 우리의 생체 내 모델보다 실험 조건의 미세한 제어 체외 방법에서 사용이 느리게 진행 혈관 장애를 연구하는 것을 목표로하고 있습니다. 시험관의 기능 수축력을 측정하는 우리는 이전에 개발 한 혈관 근육 박막 (vMTFs)은 심장 혈관 조직 (5)를 설계하지만,이 방법은 상대적으로 단기 연구에 제한되어있다. 여기서는 장기 측정 이전 vMTF 기술을 확장 기판 개질 기술을 제시한다.
혈관 내피 세포는 전체 기능에도 중요하지만, 설계 라멜라 동맥 혈관의 변화를 평가하는데 유용한 모델 시스템을 제공하는질병의 진행 동안 수축. 기능적인 혈관 질환 조직 모델 구조 및 동맥 라멜라의 기능을 모두 엔지니어, 용기의 기본 수축 부, 고 충실도로 효과적으로 요약되어야한다. 동맥 라멜라 6 엘라스틴의 시트로 구분 수축 혈관 평활근 세포의 동심, 원주 방향으로 정렬 시트입니다. 폴리 디메틸 실록산 (PDMS) 기판 상에 세포 외 기질 (ECM) 단백질의 마이크로 콘택트 프린팅 이전 심혈관 조직 5,7-10 정렬 모방 조직 조직 유도 신호를 제공하기 위해 사용되어왔다. 그러나 조직은 만성 연구에서 자신의 적용을 제한, 문화 3-4 일 후에 무결성을 잃을 수 미세 접촉 인쇄를 사용하여 패턴. 이 프로토콜은 새로운 미세 증착 기술로 기존 미세 접촉 인쇄 기술로 대체함으로써이 문제에 대한 해결책을 제공한다.
genipin와 f와 Genchi 등. 수정 된 PDMS 기판운드, 문화 (11)에 1 개월 심근 세포의 생존을 연장. 여기서는 PDMS에 패터닝 된 혈관 평활근 세포의 배양을 확장하는 유사한 접근법을 사용한다. Genipin, 치자 나무 열매의 천연 가수 유도체는 유사한 가교제 및 조직 복구 12,13 및 ECM 수식 (14)의 분야에서 생체 재료로서의 사용이 증가에 비해 인해 비교적 낮은 독성 기판 변형위한 바람직한 후보 15. 이 프로토콜에서, 피브로넥틴은 이전 미세 접촉 인쇄 방법에서와 같이, 셀 안내 큐로서 이용되고; 그러나 genipin 피브로넥틴은 패터닝 이전에 PDMS 기판 상에 증착된다. 세포가 패턴 매트릭스 저하로 따라서, 첨부 된 혈관 평활근 세포에서 새로 합성 된 ECM은 genipin 코팅 된 PDMS 기판에 바인딩 할 수 있습니다.
이 프로토콜은 두 단계 genipin과 ECM 증착을위한 미세 유체 전달 장치를 이용한다. 미세 유체 장치 모방 microco의 디자인이전의 연구 (16)에서 설계 동맥 박편에 사용 ntact 인쇄 패턴. 따라서,이 프로토콜이 성공적으로 구조 및 생체 동맥 박편의 수축 기능에 고도로 정렬 요점을 되풀이 동맥 라멜라 모방을 수득 할 것으로 예상. 우리는 또한 genipin 시험관 혈관 질환 모델에서 장기에 적합한 기판 수정 화합물 확인하기 위해 조직의 수축력을 평가.
Protocol
Representative Results
Discussion
여기서 우리는 만성 혈관 질환 경로 1,23,24의 확장 된 실험 시간은 전형적인 수 있도록 이전에 개발 된 vMTF 기술을 기반으로 구축하는 프로토콜을 제시한다. 이를 위해, 우리는 이전 MTF 수축력 실험에 사용하기위한 개선 된 혈관 조직 생존력과 엔지니어링 동맥 박편을 수득 미세 증착 기술을 이용하여 PDMS 기판 (11)의 장기간의 작용을 제공하는 것으로 나타났다 genipin를 미세 패턴. ?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.
Materials
| Coverslip staining rack | Electron Microscopy Sciences | www.emsdiasum.com/ | 72239-04 | Alternative coverslip rack may be used |
| Microscope cover glass – 25 mm | Fisher Scientific, Inc. | www.fishersci.com | 12-545-102 | Alternative brand and size may be used; Microscope slides may also be substituted as substrate base |
| Poly(N-iso-propylacrylamide) (PIPAAm) | Polysciences, Inc. | www.polysciences.com/ | #21458 | Sigma-Aldrich makes an alternate compound, but we have not tested it for use with this protocol; Compound gives strong odor, use proper ventilation |
| 1-butanol | Sigma-Aldrich | www.sigmaaldrich.com | 360465 | Hazard: flammable (store stock solution in flammable cabinet); flash point is 37 °C, avoid heating; alternative product may be used |
| Spincoater | Specialty Coating Systems, Inc. | www.scscoatings.com | SCS G3P8 Model; Alternative brand and/or model may be used | |
| Polydimethylsiloxane (PDMS) | Ellsworth Adhesives (Dow Corning) | www.ellsworth.com | 184 SIL ELAST KIT 0.5KG | Alternative distributor may be used |
| Fluorescent microbeads | Polysciences, Inc. | www.polysciences.com/ | 17151 | Alternative brand and/or larger size may be used |
| Silicon wafers | Wafer World, Inc. | www.waferworld.com | 2398 | Alternative brand and/or size may be used |
| Photoresist | MicroChem Corp. | www.microchem.com | SU-8 3025 allows 20-25-µm feature height | |
| Contact mask aligner | Suss MicroTec | www.suss.com | MA6 contact mask aligner; alternative brand and/or model may be used for wafer exposure | |
| Developer | MicroChem Corp. | www.microchem.com | SU-8 Developer; Hazard: flammable | |
| Tridecafluro-trichlorosilane | UCT Specialties, Inc. | www.unitedchem.com | T2492 | Silane for non-stick coating of patterned silicon wafers (CAUTION: Tridecafluro-trichlorosilane is a flammable and corrosive liquid. Proper personal protective equipment and local exhaust is necessary for use. ) |
| Surgical biopsy punch | Integra LifeSciences Corp. | www.miltex.com | 33-31AA-P/25 | Alternative brand and/or size may be used |
| Genipin | Cayman Chemical | www.caymanchem.com | 10010622 | Sigma-Aldrich (G4796-25MG) makes an alternate compound, but we have not tested it for use with this protocol |
| 1X phosphate buffered saline | Mediatech, Inc. | www.cellgro.com | 21-031-CV | Alternative brand may be used |
| Fibronectin | Corning, Inc. | www.corning.com | 356008 | Sigma-Aldrich (F1056) makes an alternate compound, but we have not tested it for use with this protocol |
| Penicillin/streptomycin | Life Technologies, Inc. | www.lifetechnologies.com | 15140-122 | Alternative brand and/or size may be used, as long as concentration is the same |
| Umbillical artery smooth muscle cells | Lonza | www.lonza.com | CC-2579 | Alternative cell types may be used for alternative applications. Media should be modified accordingly |
| Tyrode's solution components | Sigma-Aldrich | www.sigmaaldrich.com | various | Alternative brand may be used for mixing solution |
| Stereomicroscope | Zeiss | www.zeiss.com | 4350020000000000 | SteREOLumar V12; Alternative brand/type of stereomicroscope may be used |
| Temperature-controlled platform | Warner Instruments | www.warneronline.com | 641659; 640352; 641922 | |
| Endothelin-1 | Sigma-Aldrich | www.sigmaaldrich.com | E7764-50UG | Alternative amount may be purchased, as long as treatment concentration is maintained |
| HA-1077 | Sigma-Aldrich | www.sigmaaldrich.com | H139-10MG | Alternative amount may be purchased, as long as treatment concentration is maintained |
Referencias
- Humphrey, J. D., Baek, S., Niklason, L. E. Biochemomechanics of cerebral vasospasm and its resolution: I. A new hypothesis and theoretical framework. Ann. Biomed. Eng. 35, 1485-1497 (2007).
- Hald, E. S., Alford, P. W. Smooth muscle phenotype switching in blast traumatic brain injury-induced cerebral vasospasm. Transl. Stroke Res. 5, 385-393 (2014).
- Olivetti, G., Anversa, P., Melissari, M., Loud, A. V. Morphometry of medial hypertrophy in the rat thoracic aorta. Lab. Invest. 42, 559-565 (1980).
- , Atherosclerosis. Nature. 407, 233-241 (2000).
- Alford, P. W., Feinberg, A. W., Sheehy, S. P., Parker, K. K. Biohybrid thin films for measuring contractility in engineered cardiovascular muscle. Biomaterials. 31, 3613-3621 (2010).
- Rhodin, J. A. G., ed, B. e. r. n. e. ,. R. .. ,. Architecture of the vessel wall. Physiol. Rev. , (1979).
- Balachandran, K., et al. Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve. Proc. Natl. Acad. Sci. U. S. A. 108, 19943-19948 (2011).
- Grosberg, A., Alford, P. W., McCain, M. L., Parker, K. K. Ensembles of engineered cardiac tissues for physiological and pharmacological study: heart on a chip. 11, 4165-4173 (2011).
- Alford, P. W., Nesmith, A. P., Seywerd, J. N., Grosberg, A., Parker, K. K. Vascular smooth muscle contractility depends on cell shape. Integr. Biol. (Camb). 3, 1063-1070 (2011).
- Win, Z., et al. Smooth muscle architecture within cell-dense vascular tissues influences functional contractility). Integr. Biol. (Camb). , (2014).
- Genchi, G. G., et al. Bio/non-bio interfaces: a straightforward method for obtaining long term PDMS/muscle cell biohybrid constructs). Colloid Surface B. 105, 144-151 (2013).
- Fessel, G., Cadby, J., Wunderli, S., van Weeren, R., Snedeker, J. G. Dose- and time-dependent effects of genipin crosslinking on cell viability and tissue mechanics – Toward clinical application for tendon repair. Acta Biomater. , (2013).
- Lima, E. G., et al. Genipin enhances the mechanical properties of tissue-engineered cartilage and protects against inflammatory degradation when used as a medium supplement. J. Biomed. Mater. Res. A. 91, 692-700 (2009).
- Madhavan, K., Belchenko, D., Tan, W. Roles of genipin crosslinking and biomolecule conditioning in collagen-based biopolymer: Potential for vascular media regeneration. J. Biomed. Mater. Res. A. , (2011).
- Satyam, A., Subramanian, G. S., Raghunath, M., Pandit, A., Zeugolis, D. I. In vitro evaluation of Ficoll-enriched and genipin-stabilised collagen scaffolds. J. Tissue Eng. Regen. Med. , (2012).
- Alford, P. W., et al. Blast-induced phenotypic switching in cerebral vasospasm. Proc. Natl. Acad. Sci. U. S. A. 108, 12705-12710 (2011).
- Song, H., Tice, J. D., Ismagilov, R. F. A microfluidic system for controlling reaction networks in time. Angew. Chem. Int. Ed. Engl. 42, 768-772 (2003).
- Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3, 335-373 (2001).
- Hald, E. S., Steucke, K. E., Reeves, J. A., Win, Z., Alford, P. W. Long-term vascular contractility assay using genipin-modified muscular thin films. Biofabrication. 6, 045005 (2014).
- Han, M., Wen, J. K., Zheng, B., Cheng, Y., Zhang, C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am. J. Physiol. Cell Physiol. 291, C50-C58 (2006).
- Feinberg, A. W., et al. Muscular thin films for building actuators and powering devices. Science. 317, 1366-1370 (2007).
- Volfson, D., Cookson, S., Hasty, J., Tsimring, L. S. Biomechanical ordering of dense cell populations. Proc. Natl. Acad. Sci. U. S. A. 105, 15346-15351 (2008).
- Intengan, H. D., Schiffrin, E. L. Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis. Hypertension. 38, 581-587 (2001).
- Kayembe, K. N., Sasahara, M., Hazama, F. Cerebral aneurysms and variations in the circle of Willis. Stroke. 15, 846-850 (1984).
- McCain, M. L., Agarwal, A., Nesmith, H. W., Nesmith, A. P., Parker, K. K. Micromolded gelatin hydrogels for extended culture of engineered cardiac tissues. Biomaterials. 35, 5462-5471 (2014).
- Weir, B., Grace, M., Hansen, J., Rothberg, C. Time course of vasospasm in man. 48, 173-178 (1978).
- McCain, M. L., Sheehy, S. P., Grosberg, A., Goss, J. A., Parker, K. K. Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip. Proc. Natl. Acad. Sci. U. S. A. 110, 9770-9775 (2013).
- Agarwal, A., Goss, J. A., Cho, A., McCain, M. L., Parker, K. K. Microfluidic heart on a chip for higher throughput pharmacological studies. Lab. Chip. 13, 3599-3608 (2013).
- Huh, D., Torisawa, Y. S., Hamilton, G. A., Kim, H. J., Ingber, D. E. Microengineered physiological biomimicry: organs-on-chips. Lab. Chip. 12, 2156-2164 (2012).
- Meer, A. D., van den Berg, A. Organs-on-chips: breaking the in vitro impasse. Integr. Biol. (Camb). 4, 461-470 (2012).
