Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植
1Cardiovascular Research Institute for Intractable Disease, College of Medicine,The Catholic University of Korea, 2Division of Cardiology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine,The Catholic University of Korea, 3Division of Cardiology, Department of Internal Medicine, Uijeongbu St. Mary’s Hospital, College of Medicine,The Catholic University of Korea
Summary
干细胞治疗已成为修复心肌梗塞后受伤的心脏组织的有效策略。我们使用明胶水凝胶为干细胞移植提供最佳的体内应用，这些水凝胶能够酶交叉连接。
Abstract
目前用于预防后发性心力衰竭的心脏干细胞疗法面临的主要问题之一是受伤心肌内移植细胞的保留率和存活率低，限制了其治疗效果。最近，脚手架生物材料的使用已引起人们的关注，以改善和最大限度地提高干细胞治疗。该协议的目的是引入一种简单明了的技术，使用注射羟基丙酸（GH）水凝胶移植骨髓源性间质干细胞（MSCs）：水凝胶是心脏组织工程应用的细胞输送平台，具有现场交叉连接和高生物相容性的能力。我们提出了一个简单的方法来制造MSC加载GH水凝胶（MSC/水凝胶），并评估其生存和增殖的三维（3D）体外培养。此外，我们演示了在小鼠体内移植MSC/水凝胶的技术，描述了通过左前下降（LAD）冠状动脉结结和随后的MSC/水凝胶移植诱导心肌梗塞（MI）的外科手术。
Introduction
心脏干细胞治疗已成为心肌修复和再生的潜在方法1，2。尽管最近动物模型和临床试验取得了积极成果，但由于在梗塞的心脏组织3、4的留存率低和注射细胞存活率低，干细胞治疗心肌修复的应用有限。因此，对细胞组织工程的使用进行了深入的研究，包括注射生物材料5、心脏贴片6和细胞片7，以改善宿主心肌内的细胞保留和整合。
在生物工程心脏组织修复的各种潜在方法中，注射式水凝胶与适当的细胞类型相结合，如间质干细胞（MSCs）、胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs），是有效将细胞输送到心肌区域8、9的一个有吸引力的选择。明胶是一种众所周知的天然聚合物，与用于生物医学应用的各种生物材料相比，由于其巨大的生物相容性、可显著的生物降解性和免疫原性降低，可以用作注射基质。虽然基于明胶的注射平台有很大的潜力，但其在体内的适用性仍然有限，因为其机械刚度低，在生理环境中容易降解。
为了克服这些局限性，提出了一种由羟基丙酸组成的明胶基水凝胶的全新简单设计，用于体内应用。明胶-羟基丙烯酸（GH）结合物可在酶、马萝卜过氧化酶（HRP）存在的情况下就地交叉连接，随后在水凝胶内封装各种药物、生物分子或细胞，表明在组织工程应用10、11、12、13、14方面具有巨大潜力。此外，我们最近调查了含有封装MSC的GH水凝胶的治疗效果，并证明它们在MI后在Murine模型15中成功用于心脏修复和再生。在此协议中，我们描述了在 GH 水凝胶内封装和体外三维 （3D） 增殖的简单技术。我们还引入了一个外科手术，旨在通过冠状动脉结结和MSC加载GH水凝胶的心内移植到梗塞的心脏产生一个Murine MI模型。
Protocol
所有动物研究程序都是根据《实验室动物福利法》、《实验室动物照料和使用指南》以及韩国天主教大学医学院机构动物护理和使用委员会（IACUC）提供的啮齿动物实验指南和政策提供的。 1. MSC 和注射明胶水凝胶的准备 100 毫米培养皿中的培养 MSC，37 °C 和 5% CO2。当 MSC 增长达到 80% 的汇合时，用 DPBS 洗两次菜，并在 37 °C 下加入 1 mL 的三蛋白替代剂，持续 3 分…
Representative Results
为了有效地将 MSC 输送到梗死的心肌，本协议中使用了 图 1 中描述的 MSC 现场装载的可交叉链接水凝胶。在体内移植之前，GH水凝胶中MSC的增殖和存活通过3D体外活/死细胞染色检测（活：绿色;死：红色）证实。如图 2所示，具有代表性的图像显示了足够的 MSC 增殖，显示了 GH 水凝胶内的分支网络。此外，在第14天清楚地观察到MSC广泛的多细胞3D结构，表?…
Discussion
可注射的GH水凝胶具有巨大的潜力，在体内应用，因为他们的能力，均匀地纳入不同的治疗剂就地。此外，它们的物理和生化特性很容易根据疾病依赖性要求进行操纵。在这方面，已建议注射水凝胶，以解决目前心脏干细胞治疗的主要局限性，阻碍不良生存和细胞保留（即+10%在24小时内移植后）在受伤的心脏19，20。为了克服这一不良结果，本文所述的?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究由韩国教育部资助的韩国国家研究基金会（NRF-2018R1D1A1A02049346）基础科学研究计划支持。
Materials
| 4 % paraformaldehyde (PFA) | Intron | IBS-BP031-2 | |
| 5-0 silk suture | AILEE | SK534 | |
| 8-0 polypropylene suture | ETHICON | M8732H | |
| 8-well chamber slide | Nunc LAB-TEK | 154534 | |
| Angiocath Plus (22GA) catheter | BD Angiocath Plus | REF382423 | |
| Antibiotic-antimyocotic | Gibco | 15240-062 | |
| Centrifuge | GYROGEN | 1582MGR | |
| Confocal microscope | Zeiss | LSM 510 | |
| Cover slipe | MARIENFELD | 101242 | |
| Deluxe High Temperature Cautery kit | Bovie | QTY1 | |
| DMEM | Gibco | 11995-065 | |
| DPBS | Gibco | 14040-133 | |
| Dual-syringe | |||
| EOSIN | SIGMA-ALDRICH | HT110116 | |
| Ethanol | EMSURE | K49350783 739 | |
| FBS | Gibco | 16000-044 | |
| Fechtner conjunctiva forceps titanium | WORLD PRECISISON INSTRUMENTS | WP1820 | |
| Fluorescein isothiocyanate isomer I (FITC) | SIGMA-ALDRICH | F7250 | |
| Forcep | HEBU | HB0458 | |
| Hair removal cream | Ildong Pharmaceutical | ||
| Heating pad | Stoelting | 50300 | Homeothermic Blanket System |
| 50301 | Replacement Heating Pad for 50300 (10 X 12.5cm) | ||
| Hematoxylin | SIGMA-ALDRICH | HHS80 | |
| Horseradish peroxide (HRP; 250-330 U/mg) | SIGMA-ALDRICH | P8375 | |
| Hydrogen peroxide (H2O2; 30 wt % in H2O) | SIGMA-ALDRICH | 216763 | |
| Iodine | Green Pharmaceutical | ||
| LIVE/DEAD cell staining kit | Thermo Fisher | R37601 | |
| Mechanical ventilator | Harvard Apparatus | ||
| Micro centrifuge | HANIL | Micro 12 | |
| Micro needle holder | KASCO | 37-1452 | |
| Micro scissor | HEBU | HB7381 | |
| Microscope | OLYMPUS | SZ61 | |
| MT staining kit | SIGMA-ALDRICH | HT1079-1SET | Weigert’s iron hematoxylin solution |
| HT15-1KT | Trichrome Stain (Masson) Kit | ||
| Paraffin | LK LABKOREA | H06-660-107 | |
| PBS buffer | Gibco | 10010-023 | |
| PHK26 staining kit | SIGMA-ALDRICH | MINI26 | |
| Slide scanner | Leica | SCN400 | |
| Surgical scissor | HEBU | HB7454 | |
| Surgical tape | 3M micopore | 1530-1 | |
| Tissue cassette | Scilab Korea | Cas3003 | |
| Transducer gel | SUNGHEUNG | SH102 | |
| Trout-Barraquer needle holder curved | KASCO | 50-3710c | |
| Ultrasound system | Philips | Affiniti 50 | |
| Xylene | JUNSEI | 25175-0430 |
Referências
- Jhund, P. S., McMurray, J. J. Heart failure after acute myocardial infarction: a lost battle in the war on heart failure. Circulation. 118 (20), 2019-2021 (2008).
- Cahill, T. J., Kharbanda, R. K. Heart failure after myocardial infarction in the era of primary percutaneous coronary intervention: Mechanisms, incidence and identification of patients at risk. World Journal of Cardiology. 9 (5), 407-415 (2017).
- Cambria, E., et al. Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types. npj Regenerative Medicine. 2, 17 (2017).
- Lemcke, H., Voronina, N., Steinhoff, G., David, R. Recent Progress in Stem Cell Modification for Cardiac Regeneration. Stem Cells International. 2018, 1909346 (2018).
- Alagarsamy, K. N., Yan, W., Srivastava, A., Desiderio, V., Dhingra, S. Application of injectable hydrogels for cardiac stem cell therapy and tissue engineering. Reviews in Cardiovascular Medicine. 20 (4), 221-230 (2019).
- Gaetani, R., et al. Epicardial application of cardiac progenitor cells in a 3D-printed gelatin/hyaluronic acid patch preserves cardiac function after myocardial infarction. Biomaterials. 61, 339-348 (2015).
- Gao, L., et al. Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold. Circualtion Research. 120 (8), 1318-1325 (2017).
- Hasan, A., et al. Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction. Advanced Science. 2 (11), 1500122 (2015).
- Wu, R., Hu, X., Wang, J. Concise Review: Optimized Strategies for Stem Cell-Based Therapy in Myocardial Repair: Clinical Translatability and Potential Limitation. Stem Cells. 36 (4), 482-500 (2018).
- Lee, Y., et al. In situ forming gelatin-based tissue adhesives and their phenolic content-driven properties. Journal of Materials Chemistry B. 1 (18), 2407-2414 (2013).
- Lee, Y., Bae, J. W., Lee, J. W., Suh, W., Park, K. D. Enzyme-catalyzed in situ forming gelatin hydrogels as bioactive wound dressings: effects of fibroblast delivery on wound healing efficacy. Journal of Materials Chemistry B. 2 (44), 7712-7718 (2014).
- Lee, S. H., et al. In situ Crosslinkable Gelatin Hydrogels for Vasculogenic Induction and Delivery of Mesenchymal Stem Cells. Advanced Functional Materials. 24 (43), 6771-6781 (2014).
- Jung, B. K., et al. A hydrogel matrix prolongs persistence and promotes specific localization of an oncolytic adenovirus in a tumor by restricting nonspecific shedding and an antiviral immune response. Biomaterials. 147, 26-38 (2017).
- Kim, G., et al. Tonsil-derived mesenchymal stem cell-embedded in situ crosslinkable gelatin hydrogel therapy recovers postmenopausal osteoporosis through bone regeneration. PLoS One. 13 (7), 0200111 (2018).
- Kim, C. W., et al. MSC-Encapsulating in situ Cross-Linkable Gelatin Hydrogels To Promote Myocardial Repair. ACS Applied Bio Materials. 3 (3), 1646-1655 (2020).
- Meirelles Lda, S., Nardi, N. B. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol. 123 (4), 702-711 (2003).
- Ojha, N., et al. Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 294 (6), 2435-2443 (2008).
- Takagawa, J., et al. Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches. Journal of Applied Physiology. 102 (6), 2104-2111 (2007).
- Terrovitis, J., et al. Noninvasive Quantification and Optimization of Acute Cell Retention by In vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem Cell Delivery. Journal of the American College of Cardiology. 54 (17), 1619-1626 (2009).
- Dib, N., Khawaja, H., Varner, S., McCarthy, M., Campbell, A. Cell Therapy for Cardiovascular Disease: A Comparison of Methods of Delivery. Journal of Cardiovascular Translational Research. 4 (2), 177-181 (2011).
Tags
Keywords:
Mesenchymal Stem CellsMSCsInjectable HydrogelsIntramyocardial TransplantationMyocardial InfarctionMurine ModelCell RetentionCell SurvivalGrowth FactorsGenetic MaterialsDrugsIn Vitro ExperimentIn Vivo ExperimentCell CultureCell SuspensionTrypsinTrypan BlueCell CountingGH Conjugate SolutionHydrogen PeroxideDual Syringe
Citar este artigo
Kim, C. W., Kim, C. J., Park, E., Lee, E., Seong, E., Chang, K. Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model. J. Vis. Exp. (163), e61752, doi:10.3791/61752 (2020).