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

Masamune Morita; Yuri Ota; Kaoru Katoh; Naohiro Noda

doi:10.3791/59555

Immunology and Infection

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

Published: April 30, 2019 doi: 10.3791/59555

Masamune Morita¹, Yuri Ota^1,2, Kaoru Katoh¹, Naohiro Noda^1,2

¹Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), ²Department of Life Science and Medical Bioscience, Waseda University

Summary

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.

Abstract

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 10–30 μm 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.

Introduction

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 < 100 nm), large vesicles (LVs, <1 μm), and giant vesicles (GVs, >1 μm). 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.

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Protocol

1. Preparation of GVs Containing Bacterial Cells by the Droplet Transfer Method

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 °C.
Preparation of a lipid-containing oil solution
1. Pour 20 μL of the POPC solution and 4 μL of the biotin-PEG-DSPE solution into a glass tube (Figure 1b (i)).
2. Evaporate the organic solvent by air flow to form a lipid film and place the film in a desiccator for 1 h to completely evaporate the organic solvent (Figure 1b (ii)).
  NOTE: It is necessary to evaporate the organic solvent in a fume hood.
3. Add 200 μL of mineral oil (0.84 g/mL, Table of Materials) to the glass vial (Figure 1b (iii)).
4. Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h (Figure 1b (iii)). The final concentrations of POPC and biotin-PEG-DSPE are 1 mM and 0.002 mM, respectively.
Pre-culture of bacterial cells
1. Inoculate E. coli into 1x LB medium (1 g yeast extract, 2 g bacto tryptone, and 2 g sodium chloride in 200 mL of deionized water) from an LB plate and incubate at 37 °C for 12–14 h (overnight).
2. After incubation, collect 20 μL of the culture solution and transfer to 1.98 mL of fresh 1x LB medium, and culture the cells again for 2 h.
3. Check the optical density at 600 nm (OD₆₀₀) value of the pre-culture solution (prepared in step 1.3.2). A pre-culture solution of OD₆₀₀ = 1.0–1.5 should be used.
Preparation of the outer and inner aqueous solutions of GVs
1. Dissolve glucose in 1x LB medium to prepare an outer aqueous solution of GVs. Prepare 20 mL of a stock glucose solution (500 mM).
2. Dilute the stock glucose solution with 1x LB medium to 200 mM (Table 1).
3. Dissolve sucrose in 1x LB medium to prepare an inner aqueous solution of GVs. Prepare 20 mL of a stock sucrose solution (500 mM).
4. Mix the pre-culture solution (OD₆₀₀ = 1.0–1.5), sucrose solution (500 mM), and 1x LB medium (Table 1). The final OD₆₀₀ value of the culture solution should be 0.01–0.015 and the final sucrose concentration should be 200 mM.
  NOTE: Take care to avoid osmotic pressure. It is necessary to balance the concentration between the inner and outer aqueous solution.
Preparation of water-in-oil (W/O) droplets containing bacterial cells
1. Add 2 μL of the inner aqueous solution of GVs (prepared in step 1.4.4) to 50 μL of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) in a 0.6 mL lidded plastic tube (Figure 1b (iv)).
2. Emulsify the two components in the plastic tube by tapping the tube by hand (Figure 1b (v)).
Formation of GVs containing bacterial cells
1. Add 50 μL of the outer aqueous solution of GVs (prepared in step 1.4.2) in a 1.5 mL lidded plastic tube (Figure 1b (vi)) and gently layer 150 μL of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) on the surface of the outer aqueous solution (Figure 1b (vii)). Incubate this sample at room temperature (RT, 25 °C) for 10–15 min. Check to ensure that the interface of the oil and aqueous solutions is flat.
2. Add 50 μL of the W/O droplet solution (prepared in step 1.5.2) on the interface of the oil and aqueous solution using a pipette (Figure 1b (viii)).
3. Centrifuge the 1.5 mL lidded plastic tube (from step 1.6.2) for 10 min at 1,600 x g at RT in a desktop centrifuge (Figure 1b (ix)). After centrifugation, aspirate the oil (top layer) from the 1.5-mL lidded plastic tube using a pipette, and collect the GVs containing bacterial cells (Figure 1b (x)).

2. Preparation of a GV Observation System (Bacterial Cell Culture System)

Preparation of small vesicles (SVs) for constructing a supported bilayer membrane
1. Pour 20 μL of the POPC solution and 4 μL of the biotin-PEG-DSPE solution into a glass tube (using the same lipid composition as used for GV preparation in step 1.1).
2. Evaporate the organic solvent by air flow to form a lipid film and place this sample in a desiccator for 1 h to completely evaporate the organic solvent.
3. Add 200 μL of 200 mM glucose in 1x LB medium (the outer aqueous solution of GVs) to the glass vial.
4. Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h.
5. Prepare SVs by the extrusion method²¹ using a mini-extruder and polycarbonate membrane with 100 nm pore size.
Preparation of a handmade chamber
1. Drill a 7 mm hole with a hollow punch on a double-faced seal (10 mm x 10 mm x 1 mm).
2. Paste the double-faced seal with the hole on a cover glass (30 mm x 40 mm, thickness 0.25–0.35 mm).
Preparation of a supported bilayer membrane on the cover glass in the hole of the chamber
1. Add 30 μL of the SV solution to the hole of the chamber (prepared in section 2.2) and incubate at RT for 30 min.
2. Gently wash the hole twice with 20 μL of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.
Immobilization of GVs on the supported bilayer membrane on the cover glass in the hole of the chamber
1. Introduce 10 μL of neutravidin with the outer aqueous solution of GVs (1 mg/mL) into the hole and incubate at RT for 15 min.
2. Gently wash the hole twice with 20 μL of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.
3. Add all solution containing GVs (prepared in step 1.6.3) into the hole of the chamber and seal with a cover glass (18 mm x 18 mm, thickness 0.13–0.17 mm) (Figure 2b).
Microscopic observation of bacterial cell growth inside GVs
1. Set a microscopic heating stage system with an inverted microscope equipped with a 40x/0.6 numerical aperture (NA) objective lens with a long working distance (Figure 2b).
2. Place the chamber on the microscopic heating stage system (Figure 2b). Incubate the GVs containing bacterial cells in the chamber at a static condition for 6 h at 37 °C.
3. Capture and record microscope images of bacterial cell growth inside GVs every 30 min by using a scientific complementary metal oxide semiconductor (sCMOS) camera.

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Representative Results

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 solution) also assists the crossing of the oil-water interface of the W/O droplets. It is necessary to monitor the osmotic pressure in inner and outer aqueous solution because a slight difference in concentration between these solutions can induce deformation and collapse of GVs. A flow chart for preparing GVs containing bacterial cells by the droplet transfer method is shown in Figure 1b. By following this procedure, GVs containing single bacterial cells can be easily obtained.

To observe bacterial cell growth within GVs, an original culture system was constructed for microscopic observation (Figure 2). GVs containing bacteria were immobilized on a supported membrane surface coated with neutravidin on a cover glass (Figure 2a). This immobilization technique has enabled prolonged observation of GVs.

Typical phase-contrast microscopy images of the different sized GVs containing single bacterial cells are shown in Figure 3. In this experiment, we also obtained GVs containing bacterial cells with sizes ranging from 10 μm to 30 μm. Figure 3 shows bacterial growth at the single-cell level inside GVs with different sizes of 10.7 μm (Figure 3a) and 28 μm (Figure 3b). For both sizes of GVs, E. coli cells underwent elongation and division processes, with one or two E. coli cells growing to a very large number of cells over 6 h. Thus, E. coli cells grew stably inside the GVs.

The relative frequency of GVs containing a given number of bacterial cells is shown in Figure 4. In our experimental condition (OD₆₀₀ = 0.01–0.015), bacterial cells were encapsulated at the single cell level in approximately 10% of the obtained GVs (empty GVs were approximately 80%). The GVs encapsulated at the single cell level were approximately 50% of the GVs containing bacterial cells, as estimated from the inset of Figure 4.

Figure 1: Experimental procedures of GVs containing bacteria. (a) Scheme of GVs containing bacteria prepared by a droplet-transfer method. W/O microdroplets containing bacteria pass through a lipid monolayer interface by centrifugal force and then form a lipid bilayer membrane. (b) Flow of the synthesis of GVs containing bacteria. (i) Organic solvent containing lipids (POPC and biotin-PEG-DSPE, 100:0.2 molar ratio). (ii) Lipid film at the bottom of the glass vial. (iii) Oil solution containing lipids. (iv) Mixture of 50 μL of oil solution and 2 μL of inner aqueous solution (200 mM sucrose and 1x LB medium) containing bacterial cells. (v) Emulsification by hand tapping (over 50 times). (vi) 50 μL of the outer aqueous solution (200 mM glucose in 1x LB medium). (vii) Layering of 150 μL of oil solution on the outer aqueous solution. (viii) Layering of the W/O droplet solution. (ix) Centrifugation of the tube. (x) Precipitated GVs containing bacterial cells after aspiration of the oil. Please click here to view a larger version of this figure.

Figure 2: The observation system of bacterial cell culture inside GVs. (a) GVs are immobilized on a supported membrane through biotin–neutravidin binding on the cover glass. GVs are incubated by a heating system. (b) Picture of the observation system including a handmade chamber. Please click here to view a larger version of this figure.

Figure 3: Phase-contrast microscope images of GVs containing single bacteria cells (indicated by the black arrows). Snap-shots of bacterial cell growth inside different sized GVs. (a) Vesicle size = 10.7 μm. (b) Vesicle size = 28 μm. Please click here to view a larger version of this figure.

Figure 4: Statistical analysis of the number of encapsulated bacterial cells per GV. The relative frequencies of GVs were plotted as histograms. Inset: Magnification of the relative frequencies of GVs containing bacterial cells from single to 10< cells. A total of 235 GVs were analyzed. Please click here to view a larger version of this figure.

	Outer aqueous solution	Inner aqueous solution	Final
500 mM Glucose with LB medium	200 μL	–	200 mM
500 mM Sucrose with LB medium	–	200 μL	200 mM
1x LB medium	300 μL	295 μL	–
Pre-culture solution (OD₆₀₀ = 1.0–1.5)	–	5 μL	OD₆₀₀ = 0.01–0.015
Total volume	500 μL	500 μL

Table 1: The composition and volumes of the outer and inner aqueous solutions of GVs.

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Discussion

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 μL) of the sample solution is required to prepare the GVs. The droplet transfer method²⁰ for preparing GVs containing bacterial cells is simpler than the classical hydration²² and microfluidics methods¹⁷. For example, the classical hydration method²² is a simple and easy method for preparing GVs, but the encapsulation efficiency of materials into GVs is quite low and at least a few hundred microliters of sample is required. The recently developed cellulose paper-abetted hydration²³ and gel-assisted hydration²⁴ methods for making GVs have a high encapsulation efficiency of biomolecules compared with the classical hydration method²². Their encapsulation efficiency is as high as that of the droplet transfer technique, and it is expected that these two methods may allow the encapsulation of cells inside GVs. Moreover, the microfluidics method¹⁷ accurately encapsulates single cells inside GVs and shows a very high encapsulation efficiency of materials into GVs but requires complicated handling and techniques for fabricating microdevices and a large sample volume (at least a few milliliters) to flow the tube.

In this protocol, the stability of the oil-water interface is important for obtaining GVs containing bacterial cells (Figure 1b (vii)). To obtain many GVs, it is essential to flatten the oil-water interface. Therefore, proper preparation of the oil phase is necessary. We sonicated the oil phase for at least 1 h in a high-power ultrasonic bath (120 W) to completely dissolve the lipid molecules. It is important to layer the oil phase on the outer aqueous solution immediately after sonication (Figure 1b (vii)).

The method described here has two limitations. First, GVs often break and bacterial cells leak into the outer aqueous solution. This is because during GV formation, some W/O droplets cannot transfer through the oil-water interface and become ruptured. This is unavoidable when using the droplet transfer method²⁰. Additionally, GVs may break during observation. The stability of GVs must be improved, such as by using an artificial cytoskeleton that stabilizes GVs²⁵. Second, the number of encapsulated bacterial cells cannot be perfectly controlled. Figure 4 shows that a large number of bacterial cells were encapsulated in the GVs, and therefore, it is difficult to control the number of bacterial cells in GVs using the droplet transfer method. To control the cell number, microfluidics technology can be used.

GVs may control their inner aqueous solution more effectively than other materials (gel droplets¹²^,¹³ or W/O droplets⁵^,¹¹). For example, the aqueous conditions of the inner and outer solutions of GVs are altered by natural membrane permeability²⁶ or permeability facilitated by a membrane pore¹⁶ or transporter²⁷. In the present method, oil molecules (mineral oil in this case) remained in the membrane²⁰. The influence of the oil remaining in the membrane on the permeability of nutrients or oxygen for bacterial growth is unknown. Although we do not know the natural membrane permeability of nutrients or oxygen, we consider that the amount of nutrients or oxygen in the growth medium was sufficient for bacterial growth in the present study. The natural membrane permeability of nutrients or oxygen is very important for bacterial cell growth and is an important topic for future study. The technique for controlling permeability cannot be conducted using the culture method with gel droplets¹²^,¹³ or W/O droplets⁵^,¹¹. GVs will thus become the first choice for bacterial culture applications in a confined space.

Our bacterial culture method is a potentially new concept and tool in microbiology¹⁹ to culture unknown environmental bacteria for obtaining or analyzing their metabolic products. Moreover, our bacterial cell-containing GVs are a hybrid system of an artificial cell model (GVs) and a living cell (bacterial cells) to make a new tool for biotechnology²⁸ and bottom-up synthetic biology²⁹.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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).

Materials

Name	Company	Catalog Number	Comments
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

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Immunology and Infection