This work was supported by a Leading Initiative for Excellent Young Researchers (LEADER, No. 16812285) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, a Grant-in-Aid for Young Scientist Research (No. 18K18157, 16K21034) from Japan Society for the Promotion of Science (JSPS) to M.M., and Grant-in-Aid from MEXT to K.K. (No. 17H06417, 17H06413).

1. Preparation of GVs Containing Bacterial Cells by the Droplet Transfer Method
<ol>
	<li>Prepare lipid stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 10 mM, 1 mL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (biotin-PEG-DSPE, 0.1 mM, 1 mL) in chloroform/methanol solution (2/1, v/v) and store the stock at -20 &#176;C.</li>
	<li>Preparation of a lipid-containing oil solution
	<ol>
		<li>Pour 20 &#956;L of the POPC solution and 4 &#...

<ol>
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The authors have nothing to disclose.

Here, we describe a method for culturing bacterial cells at the single-cell level inside GVs. This simple method involves forming GVs containing bacterial cells at the single-cell level by using the droplet transfer method. Compared with other approaches for obtaining GVs containing bacterial cells, this method has two advantages: (i) it is easy to develop, and (ii) a small volume (2 &#956;L) of the sample solution is required to prepare the GVs. The droplet transfer method20 for preparing GVs con...

The culture of bacterial cells at the single-cell level has received increasing attention. Culturing bacterial cells at the single-cell level inside a confined space can elucidate bacterial functions such as phenotypic variability1,2,3,4, cell behavior5,6,7,8,9, and antibiotic resistance10,11. Because of recent advances in culture techniques, the culture of single bacteria can be achieved inside a confined space, such as in a well-chip4,7,8, gel droplet12,13, and water-in-oil (W/O) droplet5,11. To promote understanding or utilization of single bacterial cells, further technical developments of cultivation techniques are needed.
Vesicles that mimic the biological cell membrane are spherical compartments consisting of amphiphilic molecules and can hold various materials. Vesicles are classified according to size and include small vesicles (SVs, diameter &#60; 100 nm), large vesicles (LVs, &#60;1 &#956;m), and giant vesicles (GVs, &#62;1 &#956;m). SVs or LVs are commonly used as drug carriers because of their affinity to the biological cell membrane14. GVs have also been used as a reactor system for the construction of protocells15 or artificial-cells16. Encapsulation of biological cells into GVs has been reported17,18, and thus GVs show potential as a cell culture system when combined with the reactor system.
Here, along with a video of experimental procedures, we describe how GVs can be used as novel cell-culture vessels19. GVs containing bacteria were made by the droplet transfer method20 and were then immobilized on a supported membrane on a cover glass. We used this system to observe bacterial growth at the single-cell level inside GVs in real-time.

<table>
	<tbody>
		<tr>
			<td>Bactotryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Wako Pure Chemicals</td>
			<td>032-21921</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (18 &#215; 18 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>C018181</td>
			<td>thickness 0.13&#8211;0.17 mm</td>
		</tr>
		<tr>
			<td>Cover glass (30 &#215; 40 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>custom-order</td>
			<td>thickness 0.25&#8211;0.35 mm</td>
		</tr>
		<tr>
			<td>Desktop centrifuge</td>
			<td>Hi-Tech Co.</td>
			<td>ATT101</td>
			<td>swing rotor type</td>
		</tr>
		<tr>
			<td>Double-faced seal (10 &#215; 10 &#215; 1 mm)</td>
			<td>Nitoms</td>
			<td>T4613</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>AS ONE</td>
			<td>6-306-01</td>
			<td>Durham fermentation tube</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Wako Pure Chemicals</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IX-73</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako Pure Chemicals</td>
			<td>133-16771</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic heating stage system</td>
			<td>TOKAI HIT</td>
			<td>TP-110R-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Nacalai Tesque</td>
			<td>23334-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>LUCPLFLN 40&#215;/0.6 NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membranes</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>pore size 100 nm</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Zyla 4.2 plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Wako Pure Chemicals</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako Pure Chemicals</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>AS ONE</td>
			<td>ASU-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL lidded plastic tube</td>
			<td>Watson</td>
			<td>130-806C</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL lidded plastic tube</td>
			<td>Sumitomo Bakelite Co.</td>
			<td>MS4265-M</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline</td>
			<td>Avanti Polar Lipids</td>
			<td>850457P</td>
			<td>POPC</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]</td>
			<td>Avanti Polar Lipids</td>
			<td>880129P</td>
			<td>Biotin-PEG-DSPE</td>
		</tr>
	</tbody>
</table>

bacterial cell culture at the single-cell level inside giant vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We present a simple method for generating GVs containing single bacterial cells using the droplet transfer method (Figure 1). Figure 1a shows a schematic image of the precipitation of GVs containing bacteria. W/O droplets containing bacteria are transferred across the oil-water (lipid monolayer) interface by centrifugation to form GVs. The difference in density between sucrose (inner aqueous solution) and glucose (outer aqueous s...

Watch this Scientific Journal Video about Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles at JoVE.com

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

This protocol provides a means for culturing bacterial cells at the single-cell level inside giant vesicles. It's easy to develop the giant vesicles, and only a very small volume of sample solution is required to prepare the vesicles. Demonstrating the procedure will be Yuri Ota, a PhD candidate from our laboratory.Begin by adding 20 microliters of freshly prepared POPC solution and four microliters of freshly prepared biotin-PEG-DSPE solution to a glass vial. Evaporate the organic solvent by air flow until a lipid film is formed. Place the film in a desiccator for one hour to completely evaporate the organic solvent.Then add 200 microliters of mineral oil to the vial. Cover the opening of the vial with plastic paraffin film, and sonicate the vial contents in an ultrasonic bath at 120 watts for at least one hour. For a small vesicle preparation, create a lipid film, as just demonstrated, and add 200 microliters of 200 millimeter glucose in LB medium to the film.Then sonicate the film at 120 watts for at least an hour, followed by extrusion of the resulting giant vesicles into small vesicles using a mini extruder and a polycarbonate membrane with a 100-nanometer pore size. For bacterial cell pre-culture, inoculate E.coli from an LB plate into milliliters of LB medium for an overnight culture at 37 degrees Celsius. The next morning, transfer 20 microliters of the culture's supernatant to 1.98 milliliters of fresh LB medium for an additional two hours of culture.At the end of the incubation, check the optical density at 600 nanometers, or OD 600 value, of the pre-culture solution on a spectrophotometer. A pre-culture solution with an OD 600 of one to 1.5 should be used. Then mix the pre-culture solution with freshly prepared sucrose solution and fresh LB medium, according to table one.For giant vesicle formation, add 50 microliters of a freshly prepared outer aqueous solution of giant vesicles to a 1.5 milliliter lidded plastic tube. Gently layer 150 microliters of the lipid-containing oil solution over the outer aqueous solution of giant vesicles. Incubate the resulting solution for 10 to 15 minutes at room temperature, before checking to ensure that the interface of the oil and aqueous solutions is flat.For water and oil bacterial cell containing droplet formation, add two microliters of a freshly prepared inner aqueous solution of giant vesicles to 50 microliters of the sonicated lipid-containing oil solution in a 0.6 milliliter lidded plastic tube. Then, tap the tube to emulsify the two components. Next, use a pipette to add 50 microliters of water and oil droplet solution to the interface of the oil and aqueous solution, and sediment the bacterial cell-containing giant vesicles by centrifugation.Then aspirate the top oil layer and collect the giant vesicles. To prepare a handmade chamber, drill a seven-millimeter hole with a hollow punch on a 10 by 10 by one millimeter double-faced seal, then paste the double-faced seal onto a 30 by 40 millimeter cover glass. To prepare a supported bi-layer membrane, add 30 microliters of the small vesicle solution to the hole of the handmade chamber.Incubate the vesicles at room temperature for 30 minutes. At the end of the incubation, gently wash the hole two times with 20 microliters of LB medium, supplemented with 200 millimiller glucose. To immobilize giant vesicles on the supporter bi-layer membrane, introduce 10 microliters of neutravidin in outer aqueous giant vesicle solution to the hole for a 15 minute incubation at room temperature.At the end of the incubation, gently wash the hole two times with 20 microliters of LB medium, supplemented with 200 millimiller glucose, and add the entire volume of giant vesicle solution to the hole in the chamber. Then seal the hole with an 18 by 18 millimeter cover glass. To monitor bacterial growth within the giant vesicles, select the 40X objective on an inverted microscope, and place the chamber on the microscope heating stage system.Then incubate the bacterial cell-containing giant vesicles in the chamber under static conditions for six hours at 37 degrees Celsius, capturing and recording images of the bacterial cell growth every 30 minutes, with the scientific complementary metal oxide semiconductor camera. Giant vesicles containing bacterial cells typically range in size from 10 to 30 micrometers. For both sizes of giant vesicles, the E.coli cells undergo elongation and division processes, with some E.coli cells growing to a very large number of cells over the six hour observation period.The relative frequency of giant vesicles containing a single cell level is approximately 10%of the obtained giant vesicles, and the giant vesicles encapsulated at the single cell level are approximately 50%of the giant vesicles containing bacterial cells. The stability of the oil-water interface is important for obtaining giant vesicles containing bacterial cells. Take care to aspirate the oil carefully before the giant vesicle correction.After watching this video, you should have a good understanding of how to culture bacterial cells at the single cell level within individual giant vesicles. Our bacterial culture method is a new tool in microbiology for culturing unknown environmental bacteria to obtain or analyze their metabolic products.

bacterial-cell-culture-at-the-single-cell-level-inside-giant-vesicles

Research

JoVE Journal

Immunology and Infection

6.8K 조회수.  National Institute of Advanced Industrial Science and Technology (AIST). 이 프로토콜은 거대 소포 내부의 단일 세포 수준에서 세균 세포를 배양하기위한 수단을 제공합니다. 거대한 소포를 개발하는 것은 쉽고, 소포를 준비하기 위해서는 매우 적은 양의 샘플 솔루션만 필요합니다. 절차를 시연하는 것은 우리 실험실에서 박사 학위 후보인 유리 오타(유리 오타)가 될 것입니다.먼저 갓 준비된 POPC 용액 20마이크로리터와 갓 준비된 비오틴-PEG-DSP...