巨型囊泡内单细胞层的细菌细胞培养
Summary
我们演示了巨型囊泡 (GV) 内的细菌的单细胞培养。含有细菌细胞的GV通过滴转移方法制备,固定在玻璃基板上的支撑膜上,直接观察细菌生长。这种方法也可以适应其他细胞。
Abstract
我们开发了一种在巨囊(GVs)内单细胞水平上培养细菌细胞的方法。细菌细胞培养对于了解细菌细胞在自然环境中的功能非常重要。由于技术的进步,各种细菌细胞功能可以在密闭空间内的单细胞水平上显现出来。GV是由两栖脂质分子组成的球形微尺寸隔间,可以容纳各种材料,包括细胞。在这项研究中,通过滴移送法将单个细菌细胞封装到10~30μm的GV中,含有细菌细胞的GV被固定在玻璃基板上的支撑膜上。我们的方法对于观察GV内部单细菌的实时生长非常有用。我们培养大肠杆菌(大肠杆菌) 细胞作为 GV 内的模型,但这种方法可以适应其他细胞类型。我们的方法可用于微生物学、生物学、生物技术和合成生物学的科学和工业领域。
Introduction
单细胞培养受到越来越多的关注。在密闭空间内的单细胞水平上培养细菌细胞可以阐明细菌功能,如型皮变异性1,2,3,4,细胞行为5,6,7,8,9,和抗生素耐药性10,11。由于培养技术的最新进展,单细菌的培养可以在密闭的空间内实现,如在井芯片4,7,8,凝胶滴12,13,和油中水(W/O)滴5,11 。为了促进对单一细菌细胞的了解或利用,需要进一步的技术发展栽培技术。
模仿生物细胞膜的囊泡是由两栖分子组成的球形隔间,可以容纳各种材料。囊泡根据大小进行分类,包括小囊泡(SV,直径<100nm)、大囊泡(LV,1 μm)。SV或LV通常用作药物载体,因为它们与生物细胞膜14的亲和力。GV还被用作用于建造原细胞15或人工细胞16的反应堆系统。据报道,生物细胞封装到GV中已有17、18种,因此,当与反应器系统结合时,GV显示出细胞培养系统的潜力。
在这里,连同实验程序的视频,我们描述了如何GV可以用作新型细胞培养容器19。含有细菌的GV由滴转移方法20制成,然后固定在盖玻璃上的支撑膜上。我们使用该系统实时观察GV内单细胞水平的细菌生长。
Protocol
1. 通过滴转移方法制备含有细菌细胞的GV 制备1-棕榈基-2-醇-sn-甘油-3磷胆碱(POPC, 10 mM, 1 mL) 和 1,2-distearoyl-sn-甘油-3-磷乙醇胺-N-生物素(聚乙烯乙二醇)-2000] (生物素-PEG-DSPE,0.1 mM,1 mL)氯仿/甲醇溶液(2/1,v/v),并将库存储存在-20°C。 制备含脂油溶液 将 20 μL 的 POPC 溶液和 4 μL 的生物素-PEG-DSPE 溶液倒入玻璃管中(图 1b (i))。 通过气流蒸发有机?…
Representative Results
我们提出了一种使用滴转移法生成包含单个细菌细胞的GV的简单方法(图1)。图1a显示了含有细菌的GV降水的示意图。含有细菌的W/O液滴通过离心在油水(脂单层)界面上传递,形成GV。蔗糖(内部水溶液)和葡萄糖(外水溶液)之间的密度差异也有助于跨越W/O液滴的油水界面。有必要监测内水溶液和外水溶液中的渗透压力,因为这些溶液之间…
Discussion
在这里,我们描述了一种在GV内单细胞水平上培养细菌细胞的方法。这个简单的方法涉及使用液滴转移方法在单细胞水平上形成含有细菌细胞的GV。与获得含有细菌细胞的GV的其他方法相比,这种方法有两个优点:(i)易于开发,(ii)制备GV需要小体积(2μL)样品溶液。用于制备含有细菌细胞的GV的液滴转移方法20比传统的水化22和微流体方法17简单。例如,经…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了日本教育、文化、体育、科学和技术部(MEXT)的”优秀青年研究人员领导倡议”(LEADER,第16812285号)的支持,这是青年科学家研究资助(第18K18157,16K21034号)从日本科学促进会(JSPS)到M.M.,从MEXT到K.K.(第17H06417号,17H06413号)提供助学金。
Materials
| Bactotryptone | BD Biosciences | 211705 | |
| Chloroform | Wako Pure Chemicals | 032-21921 | |
| Cover glass (18 × 18 mm) | Matsunami Glass Ind. | C018181 | thickness 0.13–0.17 mm |
| Cover glass (30 × 40 mm) | Matsunami Glass Ind. | custom-order | thickness 0.25–0.35 mm |
| Desktop centrifuge | Hi-Tech Co. | ATT101 | swing rotor type |
| Double-faced seal (10 × 10 × 1 mm) | Nitoms | T4613 | |
| Glass vial | AS ONE | 6-306-01 | Durham fermentation tube |
| Glucose | Wako Pure Chemicals | 049-31165 | |
| Inverted microscope | Olympus | IX-73 | |
| Methanol | Wako Pure Chemicals | 133-16771 | |
| Microscopic heating stage system | TOKAI HIT | TP-110R-100 | |
| Mineral oil | Nacalai Tesque | 23334-85 | |
| Mini-extruder | Avanti Polar Lipids | 610000 | |
| Neutravidin | Thermo Fisher Scientific | 31000 | |
| Objective lens | Olympus | LUCPLFLN 40×/0.6 NA | |
| Polycarbonate membranes | Avanti Polar Lipids | 610005 | pore size 100 nm |
| sCMOS camera | Andor | Zyla 4.2 plus | |
| Sodium chloride | Wako Pure Chemicals | 191-01665 | |
| Sucrose | Wako Pure Chemicals | 196-00015 | |
| Ultrasonic bath | AS ONE | ASU-3D | |
| Yeast extract | BD Biosciences | 212750 | |
| 0.6 mL lidded plastic tube | Watson | 130-806C | |
| 1.5 mL lidded plastic tube | Sumitomo Bakelite Co. | MS4265-M | |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline | Avanti Polar Lipids | 850457P | POPC |
| 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] | Avanti Polar Lipids | 880129P | Biotin-PEG-DSPE |
References
- Ozbudak, E. M., Thattai, M., Kurtser, I., Grossman, A. D., van Oudenaarden, A. Regulation of noise in the expression of a single gene. Nature Genetics. 31, 69-73 (2002).
- Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., Elowitz, M. B. Gene regulation at the single-cell level. Science. 307, 1962-1965 (2005).
- Eldar, A., Elowitz, M. B. Functional roles for noise in genetic circuits. Nature. 467, 167-173 (2010).
- Hashimoto, M., et al. Noise-driven growth rate gain in clonal cellular populations. Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 3251-3256 (2016).
- Boedicker, J. Q., Vincent, M. E., Ismagilov, R. F. Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability. Angewandte Chemie International Edition. 48, 5908-5911 (2009).
- Christopher, M., Waters, B. L. B. Quorum sensing: cell-to-cell communication in bacteria. Annual Review of Cell and Developmental Biology. 21, 319-346 (2005).
- Inoue, I., Wakamoto, Y., Moriguchi, H., Okano, K., Yasuda, K. On-chip culture system for observation of isolated individual cells. Lab on a Chip. 1, 50-55 (2001).
- Wang, P., et al. Robust growth of Escherichia coli. Current Biology. 20, 1099-1103 (2010).
- Reshes, G., Vanounou, S., Fishov, I., Feingold, M. Cell shape dynamics in Escherichia coli. Biophysical Journal. 94, 251-264 (2008).
- Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L., Leibler, S. Bacterial Persistence as a Phenotypic Switch. Science. 305, 1622-1625 (2004).
- Brouzes, E., et al. Droplet microfluidic technology for single-cell high-throughput screening. Proceedings of the National Academy of Sciences of the United States of America. 106 (34), 14195-14200 (2009).
- Zengler, K., et al. Cultivating the uncultured. Proceedings of the National Academy of Sciences of the United States of America. 99 (24), 15681-15686 (2002).
- Eun, Y., Utada, A. S., Copeland, M. F., Takeuchi, S., Weibel, D. B. Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation. ACS Chemical Biology. 6, 260-266 (2011).
- Allen, T. M., Cullis, P. R. Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews. 65, 36-48 (2013).
- Szostak, J. W., Bartel, D. P., Luisi, P. L. Synthesizing life. Nature. 409, 387-390 (2001).
- Noireaux, V., Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proceedings of the National Academy of Sciences of the United States of America. 101 (51), 17669-17674 (2004).
- Tan, Y. C., Hettiarachchi, K., Siu, M., Pan, Y. R., Lee, A. P. Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles. Journal of the American Chemical Society. 128 (17), 5656-5658 (2006).
- Chowdhuri, S., Cole, C. M., Devaraj, N. K. Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse-Emulsion Technique. ChemBioChem. 17, 886-889 (2016).
- Morita, M., Katoh, K., Noda, N. Direct observation of bacterial growth in giant unilamellar vesicles: a novel tool for bacterial cultures. ChemistryOpen. 7, 845-849 (2018).
- Pautot, S., Frisken, B. J., Weitz, D. A. Production of Unilamellar Vesicles Using an Inverted Emulsion. Langmuir. 19 (7), 2870-2879 (2003).
- Hope, M. J., Bally, M. B., Webb, G., Cullis, P. R. Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochimica et Biophysica Acta (BBA) – Biomembranes. 812, 55-65 (1985).
- Tsumoto, K., Matsuo, H., Tomita, M., Yoshimura, T. Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar. Colloids and Surfaces B: Biointerfaces. 68, 98-105 (2009).
- Li, A., Pazzi, J., Xu, M., Subramaniam, A. B. Cellulose abetted assembly and temporally decoupled loading of cargo into vesicles synthesized from functionally diverse lamellar phase forming amphiphiles. Biomacromolecules. 19, 849-859 (2018).
- Weinberger, A., et al. Gel-assisted formation of giant unilamellar vesicles. Biophysical Journal. 105, 154-164 (2013).
- Kurokawa, C., et al. DNA cytoskeleton for stabilizing artificial cells. Proceedings of the National Academy of Sciences of the United States of America. 114 (28), 7228-7233 (2017).
- Nourian, Z., Roelofsen, W., Danelon, C. Triggered gene expression in fed-vesicle microreactors with a multifunctional membrane. Angewandte Chemie International Edition. 51, 3114-3118 (2012).
- Dezi, M., Di Cicco, A., Bassereau, P., Levy, D. Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents. Proceedings of the National Academy of Sciences of the United States of America. 110 (18), 7276-7281 (2013).
- Trantidou, T., Dekker, L., Polizzi, K., Ces, O., Elani, Y. Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors. Interface Focus. 8, 20180024 (2018).
- Elani, Y., et al. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules. Scientific Reports. 8, 4564 (2018).
Cite This Article
Morita, M., Ota, Y., Katoh, K., Noda, N. Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles. J. Vis. Exp. (146), e59555, doi:10.3791/59555 (2019).