多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测
Summary
这里介绍的是一种优化的方案，用于在微加工多孔设备中的固体培养基上培养分离的单个线虫。这种方法允许在个体动物的一生中监测与衰老和健康相关的各种表型，包括活动、体型和形状、运动几何形状和存活率。
Abstract
线虫秀 丽隐杆线虫 是衰老研究中最常用的模型系统之一，因为它的培养技术简单且便宜，繁殖周期快（~3天），寿命短（~3周），以及许多可用的遗传操作和分子分析工具。在 秀丽隐杆线虫中进行衰老研究的最常见方法（包括生存分析）涉及在培养皿中的固体线虫生长培养基（NGM）上一起培养数十至数百只动物的种群。虽然这种方法收集了动物种群的数据，但大多数协议不会随着时间的推移跟踪个体动物。这里介绍的是在称为WorMotels的微加工聚二甲基硅氧烷（PDMS）设备上长期培养个体动物的优化方案。每个装置允许在含有NGM的小孔中培养多达240只动物，每个孔由含硫酸铜的护城河隔离，以防止动物逃跑。本文以原始 WorMotel 描述为基础，提供了用于成型、准备和填充每个设备的详细协议，并描述了常见的技术复杂性和故障排除建议。在该协议中，有用于一致加载小容量NGM的技术，NGM和细菌食品的一致干燥，提供药物干预的选项，重复使用PDMS设备的说明和实际限制，以及即使在低湿度环境中也能最大限度地减少干燥的技巧。该技术允许在类似于培养皿中固体培养基上群体培养的标准技术的环境中纵向监测各种生理参数，包括刺激活动、未刺激活动、体型、运动几何形状、健康跨度和生存。当与自动显微镜和分析软件结合使用时，该方法与高通量数据收集兼容。最后，讨论了该技术的局限性，并将该方法与最近开发的方法进行了比较，该方法使用微托盘在固体培养基上培养分离的线虫。
Introduction
秀丽隐杆线虫 通常用于衰老研究，因为它们的生成时间短（约3天），寿命短（约3周），易于在实验室中培养，与哺乳动物的分子过程和途径的高度进化保守，以及遗传操作技术的广泛可用性。在衰老研究的背景下， 秀丽隐杆线虫 允许快速生成长寿数据和老年人口，以分析活体动物的晚年表型。进行蠕虫老化研究的典型方法包括在6cm培养皿1中手动测量固体琼脂线虫生长培养基（NGM）上以20至70只动物为一组的蠕虫群的寿命。使用年龄同步的种群可以测量整个种群中个体动物的寿命或横截面表型，但这种方法排除了随着时间的推移监测个体动物的特征。这种方法也是劳动密集型的，因此限制了可以测试的人口规模。
允许在秀丽隐杆线虫的整个生命周期中纵向监测的培养方法数量有限，每种方法都有一系列独特的优点和缺点。微流体设备，包括WormFarm2，NemaLife3和“行为”芯片4，以及5，6，7，允许随着时间的推移监测个体动物。使用多孔板在液体培养物中培养蠕虫同样允许随着时间的推移监测单个动物或少量秀丽隐杆线虫种群8，9。液体环境代表了与培养皿中固体培养基上的常见培养环境不同的环境背景，这可以改变与衰老相关的动物生理学方面，包括脂肪含量和应激反应基因的表达10，11。将这些研究与收集的大多数关于老年秀丽隐杆线虫的数据直接进行比较的能力受到潜在重要环境变量差异的限制。蠕虫畜栏12是一种在更接近于复制典型固体培养基培养物的环境中容纳个体动物的方法。蠕虫畜栏在使用水凝胶的显微镜载玻片上为每只动物包含一个密封室，可以对孤立的动物进行纵向监测。该方法使用标准明场成像来记录形态数据，例如体型和活动。然而，动物作为胚胎被放置在水凝胶环境中，在那里它们在整个生命周期中不受干扰。这需要使用有条件的无菌突变体或转基因遗传背景，这既限制了遗传筛选的能力，因为每个新的突变或转基因都需要杂交到具有条件不育的背景中，以及药物筛选的能力，因为治疗只能作为胚胎应用于动物一次。
Fang-Yen实验室开发的另一种方法允许在称为WorMotel13，14的微加工聚二甲基硅氧烷（PDMS）装置的单个孔中的固体培养基上培养蠕虫。每个装置都放置在单孔托盘中（即，尺寸与96孔板相同），并具有240个孔，由充满厌恶溶液的护城河隔开，以防止蠕虫在孔之间移动。每口井在其生命周期内可以容纳一个蠕虫。该设备被吸水聚丙烯酰胺凝胶颗粒（称为“水晶体”）包围，托盘用石蜡膜实验室薄膜密封，以保持湿度并最大限度地减少介质的干燥。该系统允许收集个体动物的健康寿命和寿命数据，而固体培养基的使用可以更好地概括绝大多数已发表的 秀丽隐杆线虫 寿命研究中动物所经历的环境，从而允许更直接的比较。最近，已经使用最初用于微细胞毒性测定15 的聚苯乙烯微托盘代替PDMS装置16开发了类似的技术。微托盘方法允许收集在固体培养基上培养的蠕虫的个性化数据，并提高了在通常会导致逃跑的条件下（例如，压力源或饮食限制）容纳蠕虫的能力，权衡是每个微托盘只能包含96只动物16，而这里使用的多孔装置最多可以容纳240只动物。
这里介绍的是用于制备多孔设备的详细方案，该方案针对板对板的一致性和并行制备多个设备进行了优化。该协议改编自方颜实验室13的原始协议。具体来说，有关于最小化污染、优化固体培养基和细菌食物源的一致干燥以及输送RNAi和药物的技术的描述。该系统可用于跟踪个人健康寿命、寿命和其他表型，例如体型和形状。这些多孔设备与现有的高通量系统兼容，可以测量寿命，这可以消除传统寿命实验中涉及的大部分体力劳动，并为大规模对单个 秀丽隐杆线虫 进行自动化、直接的寿命测量和健康跟踪提供机会。
Protocol
Representative Results
Discussion
WorMotel系统是一个强大的工具，可以随着时间的推移收集数百个孤立的 秀丽隐杆线虫 的个性化数据。继早期使用多孔装置应用于发育静止、运动行为和衰老的研究之后，这项工作的目标是优化多孔装置的制备，以便以更高的通量方式长期监测活动、健康和寿命。这项工作提供了制备多孔设备的详细协议，该协议优化了原始协议13的许多步骤，突出了可能存在技术困难的关?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH R35GM133588对GLS，美国国家医学院催化剂奖对GLS，亚利桑那州技术和研究倡议基金的支持，由亚利桑那州董事会和埃里森医学基金会管理。
Materials
| 2.5 lb weight | CAP Barbell | RP-002.5 | |
| Acrylic sheets (6 in x 4 in x 3/8 in) | Falken Design | ACRYLIC-CL-3-8/1224 | Large sheet cut to smaller sizes |
| Ampicillin sodium salt | Sigma-Aldrich | A9518 | |
| Autoclavable squeeze bottle | Nalgene | 2405-0500 | |
| Bacto agar | BD Difco | 214030 | |
| Bacto peptone | Thermo Scientific | 211677 | |
| Basin, 25 mL | VWR | 89094-664 | Disposable pipette basin |
| Cabinet style vacuum desiccator | SP Bel-Art | F42400-4001 | Do not need to use dessicant, only using as a vacuum chamber. |
| CaCl2 | Acros Organics | 349615000 | |
| Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | N2 | Wildtype strain |
| Carbenicillin | GoldBio | C-103-25 | |
| Centrifuge | Beckman | 360902 | |
| Cholesterol | ICN Biomedicals Inc | 101380 | |
| Compressed oxygen tank | Airgas | UN1072 | |
| CuSO4 | Fisher Chemical | C493-500 | |
| Dry bead bath incubator | Fisher Scientific | 11-718-2 | |
| Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | OP50 | Standard labratory food for C. elegans |
| Ethanol | Millipore | ex0276-4 | |
| Floxuridine | Research Products International | F10705-1.0 | |
| Hybridization oven | Techne | 731-0177 | Used to cure PDMS mixture, any similar oven will suffice |
| Incubators | Shel Lab | 2020 | 20 °C incubator for maintaining worm strains and 37 °C incubator to grow bacteria |
| Isopropyl ß-D-1-thiogalactopyranoside (IPTG) | GoldBio | I2481C100 | |
| K2HPO4 | Fisher Chemical | P288-500 | |
| KH2PO4 | Fisher Chemical | P286-1 | |
| Kimwipes | KimTech | 34155 | Task wipes |
| LB Broth, Lennox | BD Difco | 240230 | |
| Low melt agarose | Research Products International | A20070-250.0 | |
| MgSO4 | Fisher Chemical | M-8900 | |
| Microwave | Sharp | R-530DK | |
| Multichannel repeat pipette, 20–200 µL LTS EDP3 | Rainin | 17013800 | The exact model used is no longer sold, a similar model's catalog number has been provided |
| NaCl | Fisher Bioreagents | BP358-1 | |
| Nunc OmniTray | Thermo Scientific | 264728 | Clear polystyrene trays |
| Parafilm M | Fisher Scientific | 13-374-10 | Double-wide (4 in) |
| Petri plate, 100 mM | VWR | 25384-342 | |
| Petri plate, 60 mM | Fisher Scientific | FB0875713A | |
| Plasma cleaner | Plasma Etch, Inc. | PE-50 | |
| PLATINUM vacuum pump | JB Industries | DV-142N | |
| PolyJet 3D printer | Stratasys | Objet500 Connex3 | PolyJet 3D printing services provided by ProtoCAM (Matrial: Vero Rigid; Finish: Matte; Color: Gloss; Resolution: X-axis: 600 dpi, Y-axis: 600 dpi, Z-axis: 1600 dpi) |
| Shaking incubator | Lab-Line | 3526CC | |
| smartSpatula | LevGo, Inc. | 17211 | Disposable spatula |
| Superabsorbent polymer (AgSAP Type S) | M2 Polymer Technologies | Type S | Referred to in main text as "water crystals" |
| SYLGARD 184 Silicone Elastomer base | The Dow Chemical Company | 2065622 | |
| SYLGARD 184 Silicone Elastomer curing agent | The Dow Chemical Company | 2085925 | |
| Syringe filter (0.22 µm) | Nest Scientific USA Inc. | 380111 | |
| Syringe, 10 mL | Fisher Scientific | 14955453 | |
| TWEEN 20 | Thermo Scientific | J20605-AP | Detergent |
| Vacuum pump oil | VWR | 54996-082 | |
| VeroBlackPlus | Stratasys | RGD875 | Rigid 3D printing filament |
| Weigh boat | Thermo Scientific | WB30304 | Large enough for PDMS mixture volume |
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