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

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 variability^1,2,3,4, cell behavior^5,6,7,8,9, and antibiotic resistance^10,11. Because of recent advances in culture techniques, the culture of single bacteria can be achieved inside a confined space, such as in a well-chip^4,7,8, gel droplet^12,13, and water-in-oil (W/O) droplet^5,11. 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 reported^17,18, 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.

Protocol

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

1. 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.
2. 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.
3. 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.
4. 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.
5. 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)).
6. 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)

1. 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.
2. 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).
3. 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.
4. 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).
5. 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.

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 s...

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 con...

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