As shown in Figure 1, GV formation is initiated by preparing a lipid-oil mixture containing the desired lipid composition. The prepared lipid-oil mixture is used to form W/O droplets by mixing with the inner solution, which is composed of the desired reaction mixture, e.g., a cell-free system. The prepared droplets are layered on the outer solution. Giving a force by centrifugation induces the passage of the droplets through the interface between the oil and aqueous layers, thus forming lipid bilayer GVs in the aqueous solution. The formed GVs are collected and used for a subsequent experiment. If needed, the GVs can be washed with the outer solutions to remove the components that leaked from the broken droplets.

Although the type of lipids used for GV formation depends on the purpose of each experiment, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is generally used as a basic lipid, because of its high stability. When preparing GV using several types of lipids, the lipids dissolved in an organic solvent such as chloroform at the defined concentration are mixed to obtain the desired composition. When synthesizing membrane proteins inside GVs, an acidic phospholipid such as phosphatidylglycerol (PG) or phosphatidylserine (PS) is added to the lipid composition—for example, 10–30 mol% acidic phospholipids are used—because the negative charge on the membrane surface is important for maintaining the correct structure of membrane protein and affects to its function (Pöyry and Vattulainen, 2016). The charge is also important for the localization of peripheral membrane proteins onto the membrane surface (Furusato et al., 2018). The use of 30 mol% cholesterol makes the membrane rigid (Gracià et al., 2010; Chakraborty et al., 2020). However, it should be noted that a certain amount of cholesterol inhibits the spontaneous membrane insertion when integral membrane proteins were synthesized inside GVs or outside of liposomes (Nakamura et al., 2018; Berhanu et al., 2019). Besides cholesterol, a physiological concentration of diacylglycerol also inhibits the spontaneous insertion (Nakamura et al., 2018). Fluorescent lipids or hydrophobic dyes, such as rhodamine-phosphatidylethanolamine (PE), NBD-PE, or Nile Red, are used for labeling GV membranes. Moreover, polyethylene glycol (PEG)-lipids (2–5 KDa in size) are used to avoid the adhesion of GVs. Chemically modified unnatural lipids are also used for experimental purposes (Kurihara et al., 2011; Bhattacharya et al., 2019). Conversely, natural lipids extracted from the membranes of various organisms, such as soybean or egg yolk, are not suitable for the formation of stable GV by this method, whereas they are applicable for the electroformation method (Méléard et al., 2009) or for preparation of small-size liposomes (<200 nm in diameter). Examples of lipid compositions reported in recent artificial cell studies are shown in Table 1.

The inner solution corresponds to the internal aqueous phase of GVs. Regarding the inner solution, any component can be encapsulated in GVs, except hydrophobic molecules and detergents. For example, by adding purified proteins into the inner solution, various enzymatic reactions or structural formations in the GVs can be generated, e.g., polymerase chain reaction (Oberholzer et al., 1995), transcription (Altamura et al., 2021), actin polymerization (Lee et al., 2018; Litschel et al., 2021), Min oscillation (Litschel et al., 2018; Yoshida et al., 2019), etc. In more advanced applications, the encapsulation of a cell-free system reconstituted with purified multi-components involved in transcription and translation (Shimizu et al., 2001) or a cell extract from a certain organism such as Escherichia coli (Noireaux and Liu, 2020) allows the synthesis of desired proteins from the genes of interest. This technology has an advantage, particularly for the synthesis of membrane proteins (Furusato et al., 2018; Berhanu et al., 2019). In general, the purification of membrane proteins requires the use of a detergent that dissolves the cell membrane to isolate the proteins. The purified membrane protein sample contains a detergent, therefore it cannot be encapsulated inside GVs, although there are some exceptions (Yanagisawa et al., 2011; Altamura et al., 2017). To solve this problem, synthesizing membrane proteins inside GVs is a rational approach and allows the expression of biochemical functions on the GV membranes. This approach is depending on the phenomenon of spontaneous membrane insertion of nascent membrane proteins (Nishiyama et al., 2006). It should be noted that, when a certain amount of cholesterol or diacylglycerol is included in the composition of the GV membrane, this spontaneous insertion does not occur. The limitation of this approach is that not all membrane proteins can be integrated into the lipid membrane in a native form (Berhanu et al., 2019). For example, if a membrane protein has a large hydrophilic domain at the outside of the GV membrane (the opposite side of the translating ribosome), the spontaneous insertion with the correct membrane orientation of the protein does not occur.

In addition to purified proteins and cell-free systems, small-sized liposomes with a diameter of <200 nm can also be encapsulated, presenting the vesicle-in-vesicle structure (Lee et al., 2018; Berhanu et al., 2019; Altamura et al., 2021). This technique can mimic intracellular organelles. Interestingly, by coupling with a cell-free system that synthesizes a membrane protein, it is possible to localize the synthesized membrane proteins onto the liposome membrane (Berhanu et al., 2019). The membrane localization of the protein can be oriented using cholesterol (Nakamura et al., 2018), i.e., when cholesterol is used in the GV membrane but not in the liposome membrane, a large part of the synthesized membrane proteins are localized to the internal liposomes. The encapsulated liposome organelle is also useful for generating a proton gradient between the GV lumen and the liposome inside (Berhanu et al., 2019).

To mimic intracellular molecular crowding, Ficoll PM70 (Furusato et al., 2018; Berhanu et al., 2019) or bovine serum albumin (Fujiwara and Yanagisawa, 2014) is often encapsulated within GVs. This molecular crowding effect is essential for various types of molecular assembly and, especially, for the membrane localization of peripheral membrane proteins in association with a negative charge on the membrane surface.

In any case, the droplet transfer method generally uses sucrose in the inner solution to make it heavier than the glucose-containing outer solution, while maintaining equal osmotic pressure. In some experiments, sucrose may have a side effect on the internal reaction. In such a case, OptiPrep, a density gradient medium, is often used instead of sucrose (Van de Cauter et al., 2021).

An example of the inner solution used for the encapsulation of a commercial cell-free kit (PURE system) in GVs is shown in Table 2.

The outer solution should be made when preparing the inner solution or in advance. The composition of the outer solution should be same or very similar to that of the inner solution, except reactive molecules and genes, i.e., enzymes and DNAs. The osmolality of the outer solution should be adjusted by changing the concentration of glucose, or alternative material, to be the same as the inner osmotic pressure. For the use of cell-free systems, the basic buffer composition and amino acids should be maintained, whereas a tRNA mixture and NTPs are removed. Note that the outer solution should be prepared from a double-concentrated solution, as the final solution can be diluted with the glucose solution (see an example in Table 3). The prepared outer solution can also be used for washing GVs after their formation.

The prepared W/O droplets are transferred to the aqueous phase by giving a force from the top to the bottom of the microtube by centrifugation (Figure 1). Although a large number of the droplets are broken when they pass through the interface, successful droplets form a lipid bilayer membrane, resulting in the generation of GVs. Usually, after centrifugation, white debris appears at the interface. This has to be removed entirely together with the oil phase. The formed GVs appear at the bottom of the tube because those are containing sucrose, which is heavier than glucose in the outer solution. A portion of the bottom fraction is collected carefully and transferred into a fresh tube for further experimentation. GVs can be washed with the same outer solution. Additional reagents can be supplied to the outer solution as needed while paying attention to the change in osmotic pressure. For example, ATP can be supplied to the exterior of the GVs at the same concentration as in the internal cell-free system, considering the possible leakage of ATP from the inside.

To make the component solutions of artificial cells portable and easy to store, the mixed inner cell-free reaction solution (without DNA) and the outer solution were lyophilized. These are used to make a ready-to-use kit together with the dried lipid film and mineral oil. This enabled us to generate artificial cells within 30 min starting from the preparation of the lipid-oil to the formation of artificial cells. The successful protein synthesis using the lyophilized and rehydrated cell-free components has been reported previously (Hunt et al., 2017; Lee et al., 2020; Yang et al., 2021). Therefore, it is possible to ship the samples at ambient temperature while avoiding moisture. This has a great advantage for working outside the laboratory or exchanging materials between laboratories.

• Glass tube: diameter, 10 mm; length, 50 mm (Maruemu Corp., cat. #0407-03)

• Chloroform (CHCl₃) (Fujifilm, cat. #038-02606). CAUTION: the vapor and liquid forms of chloroform are toxic

• Phospholipids (e.g., POPC or POPG) are dissolved in CHCl₃ at 100 mM in a glass vial and briefly processed with bath sonication.

• Aluminum block heating bath (e.g., TITEC Corp., cat. #DTU-2BN)

GVs encapsulating a cell-free system (PURE system) and DNA of gfp were prepared by following the protocol described herein (Figure 2), using the outer solution described in Table 3. The resulting GVs were incubated at 37°C for 1–3 h to perform protein synthesis inside. Before incubation, 20 ng/μL of RNaseA were added to the exterior of the GVs to eliminate possible protein synthesis outside the GVs. ATP was also added to the outside of the GVs to prevent the leakage of the encapsulated ATP. The reacted GVs were observed by a confocal microscope equipped with a differential interference contrast unit. Using a 488 nm laser, we observed the synthesized green fluorescent protein (GFP) within the GVs consist of 50 mol% POPC and 50 mol% POPG (Figure 3A ).

A membrane protein was also synthesized using the same method by simply replacing the DNA. PlsY (glycerol-3-phosphate acyltransferase) is an integral membrane protein and was synthesized inside GVs. The gene encoding PlsY was fused to the GFP gene at the C terminus of the plsY DNA to visualize the product by microscopy. Note that modification of the N terminus of membrane proteins should be avoided because it may affect the co-translational spontaneous membrane localization of the proteins. Figure 3B shows successful protein synthesis in GVs and their localization onto the GV membrane.

Protein synthesis inside GVs was again performed using the lyophilized and rehydrated PURE system. All of the components of the reaction mixture including sucrose were mixed and lyophilized. After the rehydration of the PURE system, DNA encoding GFP was added and encapsulated inside GVs. The observed result was the same as that observed using the normal PURE system (Figure 3C).