用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水
1Department of Mechanical Engineering,Pohang University of Science and Technology, 2School of Interdisciplinary Bioscience and Bioengineering,Pohang University of Science and Technology, 3Asan Institute for Life Science,University of Ulsan College of Medicine and Asan Medical Center, 4Division of Hepato-Biliary and Pancreatic Surgery, Department of Surgery,University of Ulsan College of Medicine and Asan Medical Center, 5Department of Creative IT Engineering,Pohang University of Science and Technology
Summary
去细胞化细胞外基质（dECM）可以提供合适的微环境线索，以在工程结构中重述目标组织的固有功能。本文阐述了胰腺组织去细胞化、胰腺组织衍生dECM生物油墨评价以及利用生物印刷技术生成3D胰腺组织构造的方案。
Abstract
胰腺胰岛移植是1型糖尿病患者有低血糖和继发性并发症的一种有前途的治疗方法。然而，胰岛移植仍然有几个限制，如移植胰岛由于贫困胰岛移植和恶劣环境而存活率低。此外，从人类多能干细胞中分化的胰岛素生成细胞具有有限的分泌足够的激素的能力，这些激素可以调节血糖水平;因此，迫切需要通过培养具有适当微环境线索的细胞来改善成熟。在本文中，我们阐明了制备胰腺组织衍生的细胞外基质 （pdECM） 生物墨水的协议，以提供一个可增加胰腺胰岛葡萄糖敏感性的有益微环境，然后描述使用基于微糊精的生物印版技术生成3D胰腺组织构建的过程。
Introduction
最近，胰腺胰岛移植被认为是对1型糖尿病患者的一种有前途的治疗方法。该手术的相对安全性和最小侵入性是这种治疗1的很大优势。然而，它有一些限制，如分离胰岛的低成功率和免疫抑制药物的副作用。此外，由于恶劣的环境2，移植后移植胰岛的数量稳步减少。各种生物相容性材料，如藻酸盐、胶原蛋白、聚（乳胶共甘油酸）或聚乙烯乙二醇（PEG）已应用于胰岛移植，以克服这些困难。
3D细胞打印技术以其巨大的潜力和高性能在组织工程中不断涌现。不用说，生物油墨被称为提供合适的微环境，并促成印刷组织结构中细胞过程改善的重要成分。大量的剪切变薄水凝胶，如纤维蛋白、藻酸盐和胶原蛋白被广泛用作生物油墨。然而，与原生组织3中的细胞外基质（ECM）相比，这些材料表现出缺乏结构、化学、生物和机械的复杂性。微环境提示，如胰岛和ECM之间的相互作用，是增强胰岛功能的重要信号。脱细胞化ECM（dECM）可以重新创建各种ECM成分的组织特异性组合物，包括胶原蛋白、糖氨基甘油（GAG）和糖蛋白。例如，保留其周围E意象带的初级胰岛（例如，I型、III型、IV型、V型和VI型胶原蛋白、拉米宁和纤维素）表现出低细胞凋亡和更好的胰岛素敏感性，这表明组织特异性细胞-基质相互作用对于增强其与原始组织4类似的功能能力非常重要。
在本文中，我们阐明了制备胰腺组织衍生的去细胞外基质（pdECM）生物油墨的方案，为增强胰腺胰岛的活性和功能提供有益的微环境提示，然后使用基于微糊精的生物印刷技术生成3D胰腺组织结构的过程（图1）。
Protocol
猪胰腺组织是从当地的屠宰场收集的。动物实验得到了韩国首尔亚山医学中心机构动物护理和使用委员会（IACUC）的批准。 1. 组织去细胞化 准备脱细胞化的解决方案。注：所有溶液制剂中使用的1倍磷酸盐缓冲盐水（PBS）通过向10x PBS中加入蒸馏水稀释。 对于 1% Triton-X 100 溶液，使用磁搅拌棒在 150 rpm 下搅拌 6 小时，在 900 mL 的 1x PBS 中溶解 100 mL 的 100% Triton-X 10…
Representative Results
胰腺组织的去细胞化我们开发了制备 pdECM 生物墨水的工艺，以提供胰腺组织特定的微环境，以增强 3D 生物打印组织结构中的胰岛功能（图 2A）。脱细胞化过程后，97.3%的dsDNA被去除，胶原蛋白和GAG等代表性ECM成分与原生胰腺组织相比分别保持在1278.1%和96.9%（图2B）。 <p class="jove_content"…
Discussion
该协议描述了pdECM生物油墨的开发以及使用3D细胞打印技术构建3D胰腺组织结构。要概括3D工程组织构造中目标组织的微环境，生物油墨的选择至关重要。在以前的研究中，我们验证了组织特异性的dECM生物油墨有利于促进干细胞分化和增殖10。与合成聚合物相比，dECM可以作为一个有利于细胞的环境，因为组织特异性的组成和结构11。因此，在dECM中主要部件的高保?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国政府资助的国立研究基金会（NRF）的生物与医疗技术开发项目（2017M3A9C6032067）和”ICT康力创意计划”（IITP-2019-2011-1-00783）的支持。由IITP（信息与通信技术规划与评估研究所）监督。
Materials
| Biological Safety Cabinets | CRYSTE | PURICUBE 1200 | |
| Deep Freezer | Thermo Scientific Forma | 957 | |
| Digital orbital shaker | DAIHAN Scientific | DH.WSO04010 | |
| Dry oven | DAIHAN Scientific | WON-155 | |
| Freeze dryer | LABCONCO | 7670540 | |
| Fridge | SANSUNG | CRFD-1141 | |
| Grater | ABM | 1415605793 | |
| Inverted Microscopes | Leica | DMi1 | |
| Microcentrifuge | CRYSTE | PURISPIN 17R | |
| Microplate reader | Thermo Fisher Scientific | Multiskan GO | |
| Mini centrifuge | DAIHAN Scientific | CF-5 | |
| Multi-Hotplate Stirrers | DAIHAN Scientific | SMHS-6 | |
| Nanodrop | Thermo Fisher Scientific | ND-LITE-PR | |
| pH benchtop meter | Thermo Fisher Scientific | STARA2110 | |
| Rheometer | TA Instrument | Discovery HR-2 | |
| Vortex Mixer | DAIHAN Scientific | VM-10 | |
| Cirurgical Instruments | |||
| Operating Scissors | Hirose | HC.13-122 | |
| Forcep | Korea Ace Scientific | HC.203-30 | |
| Materials | |||
| 1.7 mL microcentrifuge tube | Axygen | MCT-175-C | |
| 10 ml glass vial | Scilab | SL.VI1243 | |
| 40 µm cell strainer | Falcon | 352340 | |
| 5 L beaker | Dong Sung Science | SDS 2400 | |
| 50 mL cornical tube | Falcon | 352070 | |
| 500 mL beaker | Korea Ace Scientific | KA.23-08 | |
| 500 mL bottle-top vacuum filter | Corning | 431118 | |
| 500 mL plastic container | LOCK&LOCK | INL301 | |
| 96well plate | Falcon | 353072 | |
| Aluminum foil | DAEKYO | ||
| Kimwipe | Kimtech | ||
| Magnetic bar | Korea Ace Scientific | BA.37110-0003 | |
| Mortar and pestle | DAIHAN Scientific | SC.MG100 | |
| Multi-channel pipettor | Eppendorf | 4982000314 | |
| Petri Dish | SPL | 10100 | |
| pH indicator strips | Sigma-Aldrich | 1095350001 | |
| Sieve filter mesh | DAIHAN Scientific | ||
| Decellularization | |||
| 10x pbs | Hyclone | SH30258.01 | |
| 4.7% Peracetic acid | Omegafarm | ||
| 70% ethanol | SAMCHUN CHEMICALS | E0220 SAM | |
| Distilled water | |||
| IPA | SAMCHUN CHEMICALS | samchun I0348 | |
| Triton-X 100 | Biosesang | T1020 | |
| Biochemical assay | |||
| 1,9-Dimethyl-Methylene Blue zinc chloride double salt | Sigma-Aldrich | 341088 | |
| 10 N NaOH | Biosesang | S2018 | |
| Chloramine T | Sigma-Aldrich | 857319 | |
| Chondroitin sulfate A | Sigma-Aldrich | C4384 | |
| Citric acid | Supelco | 46933 | |
| Cysteine-HCl | Sigma-Aldrich | C1276 | |
| Glacial acetic acid | Merok | 100063 | |
| Glycine | Sigma-Aldrich | 410225 | |
| HCl | Sigma-Aldrich | H1758 | |
| Na2-EDTA | Sigma-Aldrich | E5134 | |
| NaCl | SAMCHUN CHEMICALS | S2097 | |
| Papain | Sigma-Aldrich | p4762 | |
| P-DAB | Sigma-Aldrich | D2004 | |
| Perchloric acid | Sigma-Aldrich | 311421 | |
| Sodium acetate | Sigma-Aldrich | S5636 | |
| Sodium hydroxide | Supelco | SX0607N | |
| Sodium phosphate(monobasic) | Sigma-Aldrich | RDD007 | |
| Toluene | Sigma-Aldrich | 244511 | |
| Bioink | |||
| Charicterized FBS | Hyclone | SH30084.03 | |
| Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | |
| Pepsin | Sigma-Aldrich | P7215 | |
| Rose bengal | Sigma-Aldrich | 198250 | |
| RPMI-1640 medium | Thermo Fisher Scientific | 11875093 | |
| Trypan Blue solution | Sigma-Aldrich | T8154 |
References
- Shapiro, A. J., Pokrywczynska, M., Ricordi, C. Clinical pancreatic islet transplantation. Nature Reviews Endocrinology. 13 (5), 268 (2017).
- Venturini, M., et al. Technique, complications, and therapeutic efficacy of percutaneous transplantation of human pancreatic islet cells in type 1 diabetes: the role of US. Radiology. 234 (2), 617-624 (2005).
- Xie, D., et al. Cytoprotection of PEG-modified adult porcine pancreatic islets for improved xenotransplantation. Biomaterials. 26 (4), 403-412 (2005).
- Sackett, S. D., et al. Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas. Scientific Reports. 8 (1), 10452 (2018).
- Kim, J., et al. 3D cell printing of islet-laden pancreatic tissue-derived extracellular matrix bioink constructs for enhancing pancreatic functions. Journal of Materials Chemistry B. 7 (10), 1773-1781 (2019).
- Yi, H. G., et al. A bioprinted human-glioblastoma-on-a-chip for the identification of patient-specific responses to chemoradiotherapy. Nature Biomedical Engineering. 1, (2019).
- Das, S., et al. Decellularized extracellular matrix bioinks and the external stimuli to enhance cardiac tissue development in vitro. Acta Biomaterialia. , (2019).
- Kim, H., et al. Shear-induced alignment of collagen fibrils using 3D cell printing for corneal stroma tissue engineering. Biofabrication. 11 (3), 035017 (2019).
- Huang, H. H., Ramachandran, K., Stehno-Bittel, L. A replacement for islet equivalents with improved reliability and validity. Acta Diabetologica. 50 (5), 687-696 (2013).
- Pati, F., et al. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink. Nature Communications. 5, 3935 (2014).
- Hussey, G. S., Dziki, J. L., Badylak, S. F. Extracellular matrix-based materials for regenerative medicine. Nature Reviews Materials. 1, (2018).
- Kim, B. S., Kim, H., Gao, G., Jang, J., Cho, D. W. Decellularized extracellular matrix: a step towards the next generation source for bioink manufacturing. Biofabrication. 9 (3), 034104 (2017).
- Gaetani, R., et al. Evaluation of different decellularization protocols on the generation of pancreas-derived hydrogels. Tissue Engineering Part C: Methods. 24 (12), 697-708 (2018).
- Gao, G., et al. Tissue engineered bio-blood-vessels constructed using a tissue-specific bioink and 3D coaxial cell printing technique: a novel therapy for ischemic disease. Advanced Functional Materials. 27 (33), 1700798 (2017).
- La, W. G., et al. Systemically replicated organic and inorganic bony microenvironment for new bone formation generated by a 3D printing technology. RSC Advances. 6 (14), 11546-11553 (2016).
- Lee, H., et al. Development of liver decellularized extracellular matrix bioink for three-dimensional cell printing-based liver tissue engineering. Biomacromolecules. 18 (4), 1229-1237 (2017).
- Choudhury, D., Tun, H. W., Wang, T., Naing, M. W. Organ-derived decellularized extracellular matrix: a game changer for bioink manufacturing?. Trends in Biotechnology. 36 (8), 787-805 (2018).
- Kurpios, N. A., et al. The direction of gut looping is established by changes in the extracellular matrix and in cell: cell adhesion. Proceedings of the National Academy of Sciences. 105 (25), 8499-8506 (2008).
- Sakai, T., Larsen, M., Yamada, K. M. Fibronectin requirement in branching morphogenesis. Nature. 423 (6942), 876 (2003).
Cite This Article
Kim, J., Kim, M., Hwang, D. G., Shim, I. K., Kim, S. C., Jang, J. Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs. J. Vis. Exp. (154), e60434, doi:10.3791/60434 (2019).