控制用于3D细胞培养的微孔退火颗粒支架中的颗粒分数
Summary
最大限度地减少颗粒支架内颗粒组分的变异性有助于可重复的实验。这项工作描述了为 体外 组织工程应用生成具有受控颗粒组分的颗粒支架的方法。
Abstract
微凝胶是微孔退火颗粒（MAP）支架的组成部分，可作为 体外 细胞培养和 体内 组织修复的平台。在这些颗粒支架中，微凝胶之间的空隙产生的先天孔隙率使细胞浸润和迁移成为可能。控制空隙分数和颗粒分数对于MAP支架设计至关重要，因为孔隙率是细胞的生物活性线索。球形微凝胶可以在微流体装置上生成，以控制尺寸和形状，然后使用防止聚合物网络破裂的方法冷冻干燥。再水化后，冻干微凝胶导致MAP支架中的受控颗粒组分。这些微凝胶冻干方法的实施导致了可重复的研究，显示了颗粒组分对大分子扩散和细胞扩散的影响。以下协议将涵盖微凝胶的制造、冻干和再水化，用于控制MAP支架中的颗粒部分，以及通过生物正交交联对微凝胶进行退火，用于 体外3D细胞培养。
Introduction
微孔退火颗粒（MAP）支架是颗粒材料的子类，其中微凝胶（μgel）构建块相互连接以形成块状多孔支架。凭借这些颗粒支架的独特微结构，由相互连接的球形微凝胶之间的空隙产生的先天孔隙率支持加速细胞浸润和迁移1。MAP支架的微凝胶构建块可以由合成和天然聚合物制成，并具有化学改性2。这里描述的方法特别强调了由透明质酸（HA）骨架组成的微凝胶的使用，该骨架改性了功能性降冰片烯（NB）手柄。HA聚合物上的NB功能手柄支持点击化学反应，用于形成微凝胶并将它们连接在一起以生成MAP支架3，4。已经采用了许多方案将微凝胶连接在一起（即退火），例如酶1，光基5，6和无添加剂点击化学3，7反应。这项工作描述了无添加剂点击化学，使用四嗪-降冰片烯逆电子需求Diels-Alder偶联来互连HA-NB微凝胶。
为了制造MAP支架，用户首先在批处理系统或微流体装置中使用反向乳液以及电流体动力喷涂，光刻或机械破碎2生成微凝胶构建块。球形HA-NB微凝胶的生产已经得到了很好的描述，并且以前使用批量乳液2和微流体液滴生成技术8，9，10，11进行了报道。在这项工作中，在流动聚焦微流体平台上生成球形HA-NB微凝胶，用于控制尺寸和形状，如前所述8，9，10。纯化后，微凝胶存在于水悬浮液中，必须浓缩以诱导卡住状态。当被卡住时，微凝胶表现出剪切稀化特性，这使得它们能够作为可注射的空间填充材料1。诱导卡住状态的一种方法是通过冻干或冷冻干燥干燥微凝胶，然后以受控体积12对干燥产物进行再水化。或者，可以通过在过滤器上离心或通过抽吸或使用吸收材料从微凝胶沉淀中手动去除缓冲液来从微凝胶浆液中除去多余的缓冲液。然而，在制作颗粒支架时，使用离心干燥微凝胶可以产生高度可变的颗粒分数和空隙分数范围12。已经描述了使用70%IPA用于聚乙二醇（PEG）微凝胶13，氟化油用于明胶甲基丙烯酰（GelMa）微凝胶14和70%乙醇用于HA微凝胶12的冻干微凝胶技术。该协议重点介绍了使用70%乙醇（标准实验室试剂）冷冻干燥球形HA微凝胶的方法，以在干燥过程中保持原始微凝胶特性。冻干的HA微凝胶可以按用户定义的重量百分比称重和再水化，以控制MAP支架12中的最终颗粒组分。
MAP支架形成的最后一步依赖于对微凝胶进行退火以创建块状多孔支架1。通过利用天然细胞外基质成分并采用生物正交退火方案，MAP支架可作为体外细胞培养和体内组织修复的生物相容性平台3。通过这些方法，MAP支架可以由HA-NB构建块制成，具有用户定义的颗粒组分，用于组织工程应用12。以下协议描述了HA-NB微凝胶的微流体生产，然后进行冻干和再水化以控制MAP支架中的颗粒部分。最后，使用生物正交化学描述了用于体外3D细胞培养实验的微凝胶退火步骤。
Protocol
1. 微流控器件制造 软光刻注意：该协议描述了de Wilson等人9的流聚焦微流体设备设计的器件制造。但是，该协议可用于SU-8晶圆上的任何器件设计。晶片可以粘在培养皿上，然后需要硅烷化以防止PDMS粘附在晶片特征15上。将聚二甲基硅氧烷（PDMS）弹性体基料与固化剂（见 材料表）以10：1的比例混合。准备大约 100 g 以用 ~…
Representative Results
该协议的目的是演示使用生物正交交联方案以及用于3D细胞培养的受控颗粒组分制备微孔退火颗粒（MAP）支架。首先，用降冰片烯侧基修饰HA，用于微凝胶形成和互连以形成MAP支架。使用这些方法，大约31%的HA重复单元用降冰片烯功能手柄成功修饰（图1A）。具有流动聚焦区域的微流体装置（图1B）显示可产生直径~50μm或~100μm的HA-NB微凝胶（图1C，D<…
Discussion
HA-NB微凝胶的微流控生产已被证明可以产生比乳液批量生产3，9具有更窄的粒径分布范围的微凝胶。本协议中描述的微凝胶使用MMP可裂解交联剂（Ac-GCRDGPQGIWGQDRCG-NH2）配制以支持材料降解。然而，HA-NB微凝胶也可以使用替代的二硫醇接头进行交联，例如二硫苏糖醇（DTT），它是不可降解的。类似地，其它光引发剂，如Irgacure 2959 2-羟基-4-（2-羟基乙?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢美国国立卫生研究院，美国国立神经系统疾病和中风研究所（1R01NS112940，1R01NS079691，R01NS094599）和国家过敏和传染病研究所（1R01AI152568）。这项工作部分是在杜克大学共享材料仪器设施（SMIF）进行的，该设施是北卡罗来纳州研究三角纳米技术网络（RTNN）的成员，该网络由美国国家科学基金会（奖励号ECCS-2025064）支持，作为国家纳米技术协调基础设施（NNCI）的一部分。作者要感谢实验室的前博士后Lucas Schirmer博士以及Ethan Nicklow在生成用于细胞培养实验的3D打印设备方面的帮助。
Materials
| 1 mL Luer-Lok syringe sterile, single use, polycarbonate | BD | 309628 | |
| 5 mL Luer-Lok syringe sterile, single use, polycarbonate | BD | 309646 | |
| Alexa Fluor 488 C5 maleimide | Invitrogen | A10254 | For synthesis of fluorescently-labeled tetrazine |
| Alexa Fluor 647 Phalloidin | Invitrogen | A22287 | For staining cell culture samples |
| Aluminum foil | VWR | 89107-726 | |
| Biopsy punch with plunger, 1.0 mm | Integra Miltex | 69031-01 | |
| Biopsy punch, 4 mm | Integra Miltex | 33-34 | |
| Blunt needle, 23 G 0.5", Non-Sterile, Capped | SAI Infusion Technologies | B23-50 | |
| Bottle-top vacuum filter, 0.22 μm | Corning | CLS430521 | |
| Calcium chloride | VWR | 1B1110 | For microgel washing buffer |
| Capillary-piston assemblies for positive-displacement pipettes, 1000 μL max. volume | Rainin | 17008609 | |
| Capillary-piston assemblies for positive-displacement pipettes, 25 μL max. volume | Rainin | 17008605 | |
| Capillary-piston assemblies for positive-displacement pipettes, 250 μL max. volume | Rainin | 17008608 | |
| Countess Cell Counting Chamber Slides | Invitrogen | C10228 | |
| Countess II FL Automated Cell Counter | Invitrogen | AMQAF1000 | |
| Centrifuge tube, 15 mL | CELLTREAT | 667015B | |
| Centrifuge tube, 50 mL | CELLTREAT | 229421 | |
| Chloroform, ACS grade, Glass Bottle | Stellar Scientific | CP-C7304 | For synthesis of fluorescently-labeled tetrazine |
| Corona plasma gun, BD-10A High Frequency Generator | ETP | 11011 | |
| CryoTube Vials, Polypropylene, Internal Thread with Screw Cap | Nunc | 368632 | |
| D1 mouse mesenchymal cells | ATCC | CRL-12424 | Example cell line for culture in MAP gels |
| DAPI | Sigma-Aldrich | D9542 | For staining cell culture samples |
| Deuterium oxide, 99.9 atom% D | Sigma-Aldrich | 151882 | For NMR spectroscopy |
| Dialysis tubing, regenerated cellulose membrane, 12-14 kDa molecular weight cut-off | Spectra/Por | 132703 | For purifying HA-NB and HA-Tet |
| Diethyl ether | VWR | BDH1121-4LPC | For synthesis of fluorescently-labeled tetrazine |
| Dimethylformamide | Sigma-Aldrich | 277056 | For synthesis of fluorescently-labeled tetrazine |
| 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) | TCI-Chemicals | D2919 | For modifying HA |
| Dithiothreitol (DTT) | Thermo Scientific | R0861 | Non-degradable dithiol linker (substitute for MMP-cleavable peptide) |
| Dulbecco's Modified Eagle's Medium (DMEM), high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture | Sigma-Aldrich | D6429-500ML | For D1 cell culture |
| EMS Paraformaldehyde, Granular | VWR | 100504-162 | For making 4% PFA |
| Ethanol absolute (200 proof) | KOPTEC | 89234-850 | |
| Fetal bovine serum (FBS) | ATCC | 30-2020 | For D1 cell culture |
| Heating Plate | Kopf Instruments | HP-4M | |
| Hemacytometer with coverglass | Daigger Scientific | EF16034F | |
| 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) | Sigma-Aldrich | H3375 | |
| Sodium hyaluronate, 79 kDa average molecular weight, produced in bacteria Streptococcus zooepidemicus, pharmaceutical grade, microbial contamination <100 CFU/g, bacterial endotoxins <0.050 IU/mg | Contipro | N/A | 79 kDa average molecular weight was used for HA-Tet synthesis, but these methods could be adapted for other molecular weights. |
| IMARIS Essentials software package | Oxford Instruments | N/A | Microscopy image analysis software |
| Infusion pump, dual syringe | Chemyx | N/A | |
| Kimwipe | Kimberly-Clark | 34120 | |
| Laboratory stand with support lab clamp | Geyer | 212100 | |
| Liquid nitrogen | Airgas | NI 180LT22 | |
| Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate | TCI-Chemicals | L0290 | |
| Lyophilizer | Labconco | N/A | Labconco FreeZone 6 plus has been discontinued, but other lab grade console freeze dryers could be used for this protocol. |
| Methyltetrazine-PEG4-maleimide | Kerafast | FCC210 | For synthesis of fluorescently-labeled tetrazine |
| 2-(4-Morpholino)ethane Sulfonic Acid (MES) | Fisher Scientific | BP300-100 | For modifying HA |
| Micro cover glass, 24 x 60 mm No. 1 | VWR | 48393-106 | |
| Microfluidic device SU8 master wafer | FlowJem | Custom design made either in-house in clean room or outsourced | |
| Mineral oil, heavy | Sigma-Aldrich | 330760 | |
| MMP-cleavable dithiol crosslinker peptide (Ac-GCRDGPQGIWGQDRCG-NH2) | GenScript | N/A | |
| 5-Norbornene-2-methylamine | TCI-Chemicals | 95-10-3 | For HA-NB synthesis |
| Packing tape | Scotch | 3M 1426 | |
| Parafilm | Bemis | PM996 | |
| PEG(thiol)2 | JenKem Technology USA | A4001-1 | For synthesis of fluorescently-labeled tetrazine |
| Penicillin-Streptomycin, 10,000 units/mL | Thermo Fisher Scientific | 15140122 | For D1 cell culture |
| Petri dish, polystyrene, disposable, Dia. x H=150 x 15 mm | Corning | 351058 | |
| Pluronic F-127 | Sigma-Aldrich | P2443 | For washing HMPs |
| Phosphate buffered saline (PBS) 1x | Gibco | 10010023 | |
| RainX water repellent glass treatment | Grainger | 465D20 | Synthetic hydrophobic treatment solution for microfluidic device treatment |
| RGD peptide (Ac-RGDSPGERCG-NH2) | GenScript | N/A | |
| Rubber bands | Staples | 112417 | |
| Sodium chloride | Chem-Impex | 30070 | For dialysis |
| Span 80 for synthesis | Sigma-Aldrich | 1338-43-8 | |
| Sylgard 184 Silicone Elastomer | Electron Microscopy Science | 4019862 | polydimethylsiloxane (PDMS) elastomer for making microfluidic devices and tissue culture devices |
| Syringe filter, Whatman Uniflo, 0.2 μm PES, 13 mm diameter | Cytvia | 09-928-066 | |
| Tetraview LCD digital microscope | Celestron | 44347 | |
| Tetrazine-amine HCl salt | Chem-Impex | 35098 | For HA-Tet synthesis |
| Triethylamine | Sigma-Aldrich | 471283 | For synthesis of fluorescently-labeled tetrazine |
| Tris(2-carboxyethyl)phosphine (TCEP) | Millipore Sigma | 51805-45-9 | |
| Triton X-100 | VWR | 97063-864 | |
| Trypan blue solution, 0.4% | Thermo Fisher Scientific | 15250061 | |
| Trypsin EDTA (0.25%), Phenol red | Fisher Scientific | 25-200-056 | For lifting adherent cells to seed in MAP gels |
| Tygon ND-100-80 Non-DEHP Medical Tubing, Needle Gauge=23, Wall Thickness=0.020 in, Internal diameter = 0.020, Outer diameter = 0.060 in | Thomas Scientific | 1204G82 | |
| UV curing system controller, LX500 LED | OmniCure | 010-00369R | |
| UV curing head, LED spot UV | OmniCure | N/A | |
| UV light meter, Traceable | VWR | 61161-386 | |
| Vacuum dessicator | Bel-Art | 08-594-15C | |
| X-Acto Z Series Precision Utility Knife | Elmer's | XZ3601W |
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Citazione di questo articolo
Anderson, A. R., Segura, T. Controlling Particle Fraction in Microporous Annealed Particle Scaffolds for 3D Cell Culture. J. Vis. Exp. (188), e64554, doi:10.3791/64554 (2022).