3D生物打印光可调水凝胶研究成纤维细胞活化
Summary
本文介绍了如何 3D 生物打印光可调水凝胶来研究细胞外基质硬化和成纤维细胞活化。
Abstract
光可调水凝胶可以响应光照在空间和时间上发生转变。将这些类型的生物材料纳入细胞培养平台并动态触发变化，例如增加微环境硬度，使研究人员能够模拟纤维化疾病进展过程中发生的细胞外基质 （ECM） 变化。本文提出了一种用于3D生物打印能够在明胶支持浴内进行两次连续聚合反应的光电可调水凝胶生物材料的方法。通过调节支持浴的pH值来适应悬浮水凝胶的自由形式可逆嵌入（FRESH）生物打印技术，以促进Michael加成反应。首先，将含有聚乙二醇-α-甲基丙烯酸酯（PEGαMA）的生物墨水与细胞可降解交联剂进行化学计量反应，形成软水凝胶。这些软水凝胶随后暴露在光引发剂和光下，以诱导未反应基团的均聚化并使水凝胶变硬。该协议涵盖水凝胶合成、3D 生物打印、光强化和终点表征，以评估 3D 结构内的成纤维细胞活化。这里介绍的方法使研究人员能够3D生物打印各种经历pH催化聚合反应的材料，并可用于设计组织稳态，疾病和修复的各种模型。
Introduction
3D生物打印是一项变革性技术，使研究人员能够精确地将细胞和生物材料沉积在3D体积内，并重建生物组织的复杂分层结构。在过去的十年中，3D生物打印的进步已经创造了跳动的人类心脏组织1，肾脏组织的功能模型2，肺内气体交换模型3，以及用于癌症研究的肿瘤模型4。嵌入式 3D 生物打印技术的发明，例如悬浮水凝胶的自由形式可逆嵌入 （FRESH） 生物打印，使得在 3D 中再现复杂的软组织结构（如肺血管5 甚至人类心脏6 ）成为可能。 FRESH 3D 生物打印有助于通过挤出到剪切稀化支撑槽中逐层打印柔软和低粘度的生物墨水。支撑浴由紧密堆积的明胶微粒等材料组成，该微粒充当宾厄姆塑料，并在打印后保持生物墨水的预期形状和结构。一旦打印的结构凝固，就可以通过将温度升高到37°C来溶解支撑浴7。
最近的一篇评论文章总结了使用FRESH技术在各种出版物中进行3D生物打印的材料。这些天然来源的材料范围从 I 型胶原蛋白到甲基丙烯酸化透明质酸，代表了几种不同的凝胶机制7。使用这种3D生物打印技术进行的大多数研究都采用静态生物材料，这些材料不会因外部刺激而改变。动态光可调水凝胶生物材料已被我们的实验室和其他实验室使用8,9,10,11,12来模拟各种纤维化疾病。与静态生物材料不同，光可调生物墨水允许创建具有较低弹性模量值的软化模型，然后进行硬化以探索细胞对微环境硬化增加的反应。
纤维化疾病的特征是细胞外基质的产生增加，可导致瘢痕形成和硬化13。组织硬化会引发阻生组织的进一步损伤和破坏，导致永久性器官损伤甚至死亡;纤维化疾病占全球死亡人数的三分之一。成纤维细胞在这种疾病状态下产生过量和异常的细胞外基质14,15。成纤维细胞增殖增加和细胞外基质沉积进一步使组织变硬并激活促纤维化正反馈回路16,17,18,19。研究成纤维细胞活化对于了解纤维化疾病至关重要。在这里，我们将人类肺动脉高压（PAH）作为一种纤维化疾病的一个例子，其中使用3D生物打印模拟血管的3D几何形状非常重要，并介绍了光可调水凝胶的动态硬化能力。多环芳烃是一种主要肺动脉压力超过正常水平并对心脏施加压力，增加人肺动脉外膜成纤维细胞 （HPAAF） 活化并使血管组织变硬的疾病16,17,18,19。光可调聚乙二醇-α-甲基丙烯酸酯 （PEGαMA） 生物墨水配方允许构建体的时间硬化，并有助于模拟健康组织和疾病进展 5,8,9,10。利用这一独特特征能够量化 HPAAF 响应 3D 微环境硬化的激活和增殖，并可能为参与该疾病的细胞机制提供有价值的见解。这里描述的协议将允许研究人员创建3D模型，以概括疾病进展或组织修复期间细胞外微环境的变化，并研究成纤维细胞活化。
Protocol
1. PEGαMA的合成与表征 注：聚乙二醇-α-甲基丙烯酸酯（PEGαMA）的合成改编自Hewawasam 等人， 并在无水分条件下进行9。 称量反应物。注：例如，称取 5 g 10 kg/mol 8 臂 PEG-羟基 （PEG-OH） 和 0.38 g 氢化钠 （NaH）（参见 材料表）。 向 250 mL 舒伦克烧瓶中加入搅拌棒，并用氩气吹扫。 将PEG-OH溶解在舒伦克烧瓶…
Representative Results
该协议描述了如何在支撑浴中3D生物打印光可调水凝胶，以创建能够动态和时间加固的结构，以研究模拟人体组织的几何形状中的成纤维细胞活化。首先，该协议解释了如何合成PEGαMA，这是该光电互导聚合物系统的骨架。核磁共振（NMR）波谱测量显示PEGαMA功能化成功率为96.5%（图1）。对于此程序，90%或更高的功能化值是可以接受的。接下来，详细介绍了如何创建空心圆柱体…
Discussion
响应受控光照的双阶段聚合反应可以通过空间和时间控制使生物材料变硬。一些研究利用这种技术来评估各种平台中的细胞-基质相互作用 5,8,9,10,11,21,22,23。更具体地说，光掩模或双光子激发方法?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢 Adam Feinberg 博士（卡内基梅隆大学）和主持 3D 生物打印开源研讨会的人员。这些人使学习FRESH生物打印技术成为可能，并构建了用于这些研究的3D生物打印机。此外，作者要感谢 Biorender.com，它被用来制作本手稿中的数字。这项工作得到了多个团体或资金来源的支持，包括罗斯社区基金会（DDH 和 CMM）、科罗拉多州肺血管疾病研究奖（DDH 和 CMM）、1941401 奖下的国家科学基金会 （CMM）、W81XWH-20-1-0037 （CMM） 下陆军部、美国国立卫生研究院国家癌症研究所 R21 CA252172 奖 （CMM）、 科罗拉多大学安舒茨医学院的卢德曼家庭女性健康研究中心（DDH 和 CMM）、美国国立卫生研究院国家心肺血液研究所获得 R01 HL080396 （CMM）、R01 HL153096 （CMM）、F31 HL151122 （DDH） 和 T32 HL072738（DDH 和 AT）奖项。
Materials
| AccuMax Radiometer/Photometer Kit | Spectronics Corporation | XPR-3000 | To measure light intensity, used for photostiffening |
| Acetic Acid | Fisher Scientific | BP2401-500 | Used during PEGaMA synthesis |
| Acetone | Fisher Scientific | A184 | Used with the cryosections |
| ActinGreen 488 ReadyProbes | Fisher Scientific | R37110 | Used for staining |
| Aluminum Foil | Reynolds | F28028 | |
| Anhydrous Tetrahydrofuran (THF) | Sigma-Aldrich | 401757-1L | Used during PEGaMA synthesis |
| Argon Compressed Gas | Airgas | AR R300 | Used during PEGaMA synthesis |
| 8 Arm Poly(ethylene glycol)-hydroxyl (PEG-OH) | JenKem Technology | 8ARM-PEG-10K | Used during PEGaMA synthesis |
| 365 nm Bandpass Filter | Edmund Optics | 65-191 | Used for photostiffening |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP9700-100 | Used during staining process |
| Buchner Funnel | Quark Glass | QFN-8-14 | Used during PEGaMA synthesis |
| Calcein AM | Invitrogen | 65-0853-39 | Used during staining process |
| Celite 545 (Filtration Aid) | EMD Millipore | CX0574-1 | Used during PEGaMA synthesis |
| Charged Microscope Slides | Globe Scientific | 1358W | |
| Chloroform-d | Sigma-Aldrich | 151823-10X0.75ML | Used to characterize PEGaMA |
| Click-iT Plus EdU Cell Proliferation Kit | Invitrogen | C10637 | Used for staining |
| 50 mL Conical Tubes | CELLTREAT | 667050B | |
| Cryogenic Safety Kit | Cole-Parmer | EW-25000-85 | |
| Cryostat | Leica | CM 1850-3-1 | |
| Dialysis Tubing | Repligen | 132105 | |
| 4’,6-Diamidino-2-Phylindole (DAPI) | Sigma-Aldrich | D9542-1MG | Used for staining |
| Diethyl Ether | Fisher Scientific | E1384 | Used during PEGaMA synthesis |
| 1,4-Dithiothreitol (DTT) | Sigma-Aldrich | 10197777001 | Bioink component |
| Dulbecco's Modified Eagle's Medium (DMEM) | Cytiva | SH30271.FS | |
| Ethyl 2-(Bromomethyl)Acrylate (EBrMA) | Ambeed Inc. | A918087-25g | Used during PEGaMA synthesis |
| Filter Paper | Whatman | 1001-090 | Used during PEGaMA synthesis |
| Freezone 2.5L Freeze Dry System | Labconco | LA-2.5LR | Lyophilizer |
| Fusion 360 | Autodesk | N/A | Software download |
| 2.5 mL Gastight Syringe | Hamilton | 81420 | Used for bioprinting |
| 15 Gauge 1.5" IT Series Tip | Jensen Global | JG15-1.5X | Used for bioprinting |
| 30 Gauge 0.5" HP Series Tip | Jensen Global | JG30-0.5HPX | Used for bioprinting |
| Goat Anti-Mouse Alexa Fluor 555 Antibody | Fisher Scientific | A21422 | Used for staining |
| Glycine | Fisher Scientific | C2H5NO2 | Used during staining process |
| Hemocytometer | Fisher Scientific | 1461 | |
| Hoechst | Thermo Scientific | 62249 | Used during staining process |
| Human Pulmonary Artery Adventitial Fibroblasts (HPAAFs) | AcceGen | ABC-TC3773 | From a 2-year-old male patient |
| Hydrochloric Acid (HCl) | Fisher Scientific | A144-500 | Used to pH adjust solutions |
| ImageJ | National Institutes of Health (NIH) | N/A | Free software download |
| ImmEdge® Pen | Vector Laboratories | H-4000 | Used during staining process |
| Incubator | VWR | VWR51014991 | |
| LifeSupport Gelatin Microparticle Slurry (Gelatin Slurry) | Advanced Biomatrix | 5244-10GM | Used for bioprinting |
| Light Microscope | Olympus | CKX53 | Inverted light microscope |
| Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate (LAP) | Sigma-Aldrich | 900889-5G | Photoinitiator used for photostiffening |
| Liquid Nitrogen | N/A | N/A | |
| LulzBot Mini 2 | LulzBot | N/A | Bioprinter adapted |
| Methacryloxyethyl Thiocarbamoyl Rhodamine B | Polysciences Inc. | 669775-30-8 | |
| 2-Methylbutane | Sigma-Aldrich | M32631-4L | |
| Microman Capillary Pistons CP1000 | VWR | 76178-166 | Positive displacement pipette tips |
| MMP2 Degradable Crosslinker (KCGGPQGIWGQGCK) | GL Biochem | N/A | Bioink component |
| Mouse Anti-Human αSMA Monoclonal Antibody | Fisher Scientific | MA5-11547 | Used for staining |
| OmniCure Series 2000 | Lumen Dynamics | S2000-XLA | UV light source used for photostiffening |
| Paraformaldehyde (PFA) | Electron Microscopy Sciences | 15710 | Used to fix samples |
| pH Meter | Mettler Toledo | FP20 | |
| pH Strips | Cytiva | 10362010 | |
| Phosphate Buffered Saline (PBS) | Hyclone Laboratories, Inc. | Cytiva SH30256.FS | |
| Pipette Set | Fisher Scientific | 14-388-100 | |
| 10 µL Pipette Tips | USA Scientific | 1120-3710 | |
| 20 µL Pipette Tips | USA Scientific | 1183-1510 | |
| 200 µL Pipette Tips | USA Scientific | 1111-0700 | |
| 1000 µL Pipette Tips | USA Scientific | 1111-2721 | |
| Poly(Ethylene Glycol)-Alpha Methacrylate (PEGαMA) | N/A | N/A | Refer to manuscript for synthesis steps |
| Poly(Ethylene Oxide) (PEO) | Sigma-Aldrich | 372773-250G | Bioink component |
| Positive Displacement Pipette | Fisher Scientific | FD10004G | 100-1000 µL |
| Potassium Hydroxide (KOH) | Sigma-Aldrich | 221473-500G | Used to pH adjust solutions |
| ProLong Gold Antifade Reagent | Invitrogen | P36930 | Used during staining process |
| Pronterface | All3DP | N/A | Software download |
| Propidium Iodide | Sigma-Aldrich | P4864-10ML | Used for staining |
| RGD Peptide (CGRGDS) | GL Biochem | N/A | Bioink component |
| Rocker | VWR | 10127-876 | |
| Rotary Evaporator | Thomas Scientific | 11100V2022 | Used during PEGaMA synthesis |
| Rubber Band | Staples | 808659 | |
| Schlenk Flask | Kemtech America | F902450 | Used during PEGaMA synthesis |
| Slic3r | Slic3r | N/A | Software download |
| Smooth Muscle Cell Growth Medium-2 (SmGM-2) BulletKit | Lonza | CC-3182 | Kit contains CC-3181 and CC-4149 components |
| Sodium Hydride | Sigma-Aldrich | 223441-50G | Used during PEGaMA synthesis |
| Sorvall ST 40R Centrifuge | Fisher Scientific | 75-004-525 | |
| Stir Bar | VWR | 58948-091 | |
| Syringe Filter | VWR | 28145-483 | Used to sterile filter solutions |
| T-75 Tissue-Cultured Treated Flask | VWR | 82050-856 | Used for cell culture work |
| Tissue-Tek Cyromold | Sakura | 4557 | |
| Tissue-Tek O.C.T Compound (OCT) | Sakura | 4583 | |
| Tris(2-Carboxyethyl) Phosphine (TCEP) | Sigma-Aldrich | C4706-2G | |
| Triton X-100 | Fisher Bioreagents | C34H622O11 | Used during staining process |
| Trypan Blue | Sigma-Aldrich | T8154-20ML | Used for cell culture work |
| 0.05% Trypsin-EDTA | Gibco | 25-300-062 | Used for cell culture work |
| Tween 20 | Fisher Bioreagents | C58H114O26 | Used during staining process |
| Upright Microscope | Olympus | BX63F | Fluorescent microscope capabilities |
| Water Bath | PolyScience | WBE20A11B | |
| 24-Well Tissue Culture Plates | Corning | 3527 |
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Citazione di questo articolo
Tanneberger, A. E., Blair, L., Davis-Hall, D., Magin, C. M. 3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation. J. Vis. Exp. (196), e65639, doi:10.3791/65639 (2023).