We thank Tomoko Yamaguchi for drawing the schematic image in Figure 1. This study was supported by the Okazaki ORION Project of the Okazaki Institute for Integrative Bioscience of theNational Institutes of Natural Sciences (NINS); by the Astrobiology Center Project (no. AB281010) of NINS; by a Grant-in-Aid for Scientific Research on Innovative Areas (Dynamical ordering of biomolecular systems for creation of integrated functions) (no. 25102008) from the Japan Society for the Promotion of Science (JSPS); by a Grant-in-Aid for Young Scientists (B) (to K.K., no. 15K17850) from JSPS; andby a Grant-in-Aid for Research Activity Start-Up (to Y.N., no. 15H06653) from JSPS. Additional support was provided by a grant from the Noguchi Institute, a grant from the Kao Foundation for Arts and Sciences, and a grant from the Kurita Water and Environment Foundation.

1. Preparation of GVs by the W/O&#160;Emulsion Centrifugation Method
<ol>
	<li>Prepare a stock solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 25 mM) and a stock solution of Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE, 0.20 mM) in chloroform and store the stock solutions at -20 &#176;C.</li>
	<li>Prepare an oil solution.
	<ol>
		<li>Form a lipid film on the inside surface of a 5&#160;ml glass vial by evaporating a mixture of the DOPC stock solution (51.0 &#181;l and the Texas Red DHPE stock solution (19.2 &#181;l) under flowing nitrogen gas.</li>
		<li>Incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin (0.86-0.89 g/cm3) to the vial. Wrap the vial in aluminum foil and incubate it at 80 &#176;C O/N at rest. The final concentrations of DOPC and Texas Red DHPE are 1.3 and 3.8 x 10&#8722;3 mM, respectively, and the DOPC:Texas Red DHPE molar ratio is 100:0.3.</li>
	</ol>
	</li>
	<li>Prepare the inner aqueous dispersion.
	<ol>
		<li>In a 1.5&#160;ml lidded microtube, mix 237.5 &#181;l of a dispersion of 1.0&#160;&#181;m nonfluorescent microspheres (2.5 vol%) and 12.5 &#181;l of a dispersion of 1.0&#160;&#181;m fluorescent microspheres (2.5 vol%); this corresponds to a 95:5 (v/v) ratio of nonfluorescent to fluorescent microspheres.</li>
		<li>Add 64 mg of sucrose followed by 125 &#181;l of Tris-buffered solution (TBS, 1 M) and 875 &#181;l of deionized water. The final volume fraction of microspheres is 0.5 vol%, and the final concentrations of Tris-HCl (pH 7.5) and sucrose are 0.1 and 0.15 M, respectively.</li>
		<li>Vortex the microtube for 30 sec and then sonicate it for 10 min.</li>
	</ol>
	</li>
	<li>Prepare the outer aqueous solution.
	<ol>
		<li>After preparation of 10 ml of a Tris-buffered solution (0.1 M Tris-HCl [pH 7.5], 0.15 M glucose) in the same procedure as inner aqueous media, place 1 ml of the solution in a 1.5&#160;ml lidded microtube. Vortex the microtube for 30 sec and then sonicate it for 10 min.</li>
	</ol>
	</li>
	<li>Prepare a w/o emulsion containing microspheres.
	<ol>
		<li>Mix 1 ml of the oil solution (liquid paraffin containing DOPC and Texas Red DHPE) with 300 &#181;l of the inner aqueous solution in a 1.5&#160;ml microtube.</li>
		<li>Emulsify the two components in the microtube by using a mechanical homogenizer (an agitator with blades that rotate at high speed) operated at 10,000 rpm for 2 min at RT.</li>
	</ol>
	</li>
	<li>Precipitate the GVs.
	<ol>
		<li>Gently layer 300 &#181;l of the w/o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 &#176;C in a 1.5&#160;ml lidded microtube. Chill the microtube for 10 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Collect the GVs.
	<ol>
		<li>Immediately after chilling the microtube, centrifuge it at 18,000 x g for 30 min. Obtain the precipitated GVs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.</li>
		<li>Dilute the precipitated GV droplet 10-100 fold by volume with the outer aqueous solution if the obtained GVs are obtained in quantities large enough to make observation difficult.</li>
	</ol>
	</li>
</ol>
2. Microscopy Observation of GVs
<ol>
	<li>Prepare microscopy specimen.
	<ol>
		<li>Place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization (chamber size 9 mm x 9 mm x 0.3 mm thick) on top of a microscope cover glass.</li>
		<li>Using a micropipette, deposit 25 &#181;l of the diluted precipitated GVs on the specimen area and immediately place another cover glass (approximately 0.15 mm thick) on top of the incubation chamber.</li>
	</ol>
	</li>
	<li>Record differential interference contrast microscopy images of the GVs.
	<ol>
		<li>Record microscopy images of the vesicles with a microscope (10X, 20X, and 40X&#160;objectives) equipped with a 12V100WHAL-L halogen lamp.</li>
	</ol>
	</li>
	<li>Conduct fluorescence microscopy observations.
	<ol>
		<li>Insert U-FBNA and U-FMCHE fluorescence mirror units into the microscope. Fit the units with 470-495&#160;nm and 565-585&#160;nm excitation filters, respectively, and with emission filters that transmit 510-550&#160;nm and 600-690&#160;nm light, respectively.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Luisi, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The emergence of life: from chemical origins to synthetic biology. , (2006).</li><li>Luisi, P. L., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The minimal cell: the biophysics of cell compartment and the origin of cell. , (2011).</li><li>Rasmussen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocells: Bridging nonliving and living matter. , (2008).</li><li>Luisi, P. L., Walde, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Giant vesicles: perspectives in supramolecular chemistry. , (2000).</li><li>Kita, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+genetic+information+with+self-encoded+replicase+in+liposomes.">Replication of genetic information with self-encoded replicase in liposomes.</a> Chem Bio Chem. 9 (15), 2403-2410 (2008).</li><li>Nomura, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+within+cell-sized+lipid+vesicles.">Gene expression within cell-sized lipid vesicles.</a> Chem Bio Chem. 4 (11), 1172-1175 (2003).</li><li>Oberholzer, T., Albrizio, M., Luisi, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction+in+liposome.">Polymerase chain reaction in liposome.</a> Chem Biol. 2 (10), 677-682 (1995).</li><li>Shohda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartment+size+dependence+of+performance+of+polymerase+chain+reaction+inside+giant+vesicle.">Compartment size dependence of performance of polymerase chain reaction inside giant vesicle.</a> Soft Matter. 7 (8), 3750-3753 (2011).</li><li>Kurihara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-reproduction+of+supramolecular+giant+vesicles+combined+with+the+amplification+of+encapsulated+DNA.">Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.</a> Nat Chem. 3 (10), 775-781 (2011).</li><li>Kurihara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recursive+vesicle-based+model+protocell+with+a+primitive+model+cell+cycle.">A recursive vesicle-based model protocell with a primitive model cell cycle.</a> Nat Comm. 6, 8352 (2015).</li><li>Walde, P., Cosentino, K., Engel, H., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+vesicles:+preparations+and+applications.">Giant vesicles: preparations and applications.</a> Chem Bio Chem. 11 (7), 848-865 (2010).</li><li>Reeves, J. P., Dowben, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+properties+of+thin-walled+phospholipid+Vesicles.">Formation and properties of thin-walled phospholipid Vesicles.</a> J Cell Physiol. 73 (1), 49-60 (1969).</li><li>Needham, D., Evans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanical+properties+of+giant+lipid+(DMPC)+vesicle+bilayers+from+20+&#176;C+below+to+10+&#176;C+above+the+liquid+crystal-crystalline+phase+transition+at+24+&#176;C.">Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 &#176;C below to 10 &#176;C above the liquid crystal-crystalline phase transition at 24 &#176;C.</a> Biochemistry. 27 (21), 8261-8269 (1988).</li><li>Oku, N., MacDonald, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+alkali+metal+chlorides+on+formation+of+giant+liposomes+by+freezing+and+thawing+and+by+dialysis.">Differential effects of alkali metal chlorides on formation of giant liposomes by freezing and thawing and by dialysis.</a> Biochemistry. 22 (4), 855-863 (1983).</li><li>Angelova, M. I., Dimitrov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+electroformation.">Liposome electroformation.</a> Far Disc Chem Soc. 81, 303-311 (1986).</li><li>Dimitrov, D. S., Angelova, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+swelling+and+liposome+formation+on+solid+surfaces+in+external+electric+fields.">Lipid swelling and liposome formation on solid surfaces in external electric fields.</a> New Trends Coll Sci. 73, 48-56 (1987).</li><li>Funakoshi, K., Suzuki, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+giant+lipid+vesicle+like+compartments+from+a+planar+lipid+membrane+by+a+pulsed+jet+flow.">Formation of giant lipid vesicle like compartments from a planar lipid membrane by a pulsed jet flow.</a> J Am Chem Soc. 129 (42), 12608-12609 (2007).</li><li>Stachowiak, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilamellar+vesicle+formation+and+encapsulation+by+microfluidic+jetting.">Unilamellar vesicle formation and encapsulation by microfluidic jetting.</a> Proc Natl Acad Sci USA. 105 (12), 4697-4702 (2008).</li><li>Ellis, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding:+obvious+but+underappreciated.">Macromolecular crowding: obvious but underappreciated.</a> Trends Biochem Sci. 26 (10), 597-604 (2001).</li><li>Ellis, R. J., Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology:+join+the+crowd.">Cell biology: join the crowd.</a> Nature. 425 (6953), 27-28 (2003).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+unilamellar+vesicles+using+an+inverted+emulsion.">Production of unilamellar vesicles using an inverted emulsion.</a> Langmuir. 19 (7), 2870-2879 (2003).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+asymmetric+vesicles.">Engineering asymmetric vesicles.</a> Proc Natl Acad Sci USA. 100 (19), 10718-10721 (2003).</li><li>Nishimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population+analysis+of+structural+properties+of+giant+liposomes+by+flow+cytometry.">Population analysis of structural properties of giant liposomes by flow cytometry.</a> Langmuir. 25 (18), 10439-10443 (2009).</li><li>Chiba, M., Miyazaki, M., Ishiwata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+lamellarity+of+giant+liposomes+prepared+by+the+inverted+emulsion+method.">Quantitative analysis of the lamellarity of giant liposomes prepared by the inverted emulsion method.</a> Biophys J. 107 (2), 346-354 (2014).</li><li>Cabr&#233;, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+division+proteins+FtsZ+and+ZipA+induce+vesicle+shrinkage+and+cell+membrane+invagination.">Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination.</a> J Biol Chem. 288, 26625-26634 (2013).</li><li>Osawa, M., Erickson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+division+by+a+simple+bacterial+division+machinery.">Liposome division by a simple bacterial division machinery.</a> Proc Natl Acad Sci USA. 110 (27), 11000-11004 (2013).</li><li>Hayashi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+morphological+control+of+tubulin-encapsulating+giant+liposomes+by+hydrostatic+pressure.">Reversible morphological control of tubulin-encapsulating giant liposomes by hydrostatic pressure.</a> Langmuir. 32 (15), 3794-3802 (2016).</li><li>Terasawa, H., Nishimura, K., Suzuki, H., Matsuura, T., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+the+fusion+and+budding+of+giant+phospholipid+vesicles+containing+macromolecules.">Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules.</a> Proc Natl Acad Sci USA. 109 (16), 5942-5947 (2012).</li><li>Natsume, Y., Toyota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetrical+polyhedral+configuration+of+giant+vesicles+induced+by+orderly+array+of+encapsulated+colloidal+particles.">Asymmetrical polyhedral configuration of giant vesicles induced by orderly array of encapsulated colloidal particles.</a> PLOS ONE. 11 (1), e0146683 (2016).</li><li>Natsume, Y., Toyota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+crystalline+pattern+for+colloidal+particles+encapsulated+in+giant+vesicles:+direct+cross-sectional+observation+of+ordered+and+disordered+phases.">Appearance of crystalline pattern for colloidal particles encapsulated in giant vesicles: direct cross-sectional observation of ordered and disordered phases.</a> Trans Mater Res Soc Jpn. 41 (2), 147-149 (2016).</li><li>Noireaux, V., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vesicle+bioreactor+as+a+step+toward+an+artificial+cell+assembly.">A vesicle bioreactor as a step toward an artificial cell assembly.</a> Proc Natl Acad Sci USA. 101 (51), 17669-17674 (2004).</li><li>Fujii, S., Matsuura, T., Sunami, T., Kazuta, Y., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evolution+of+&#945;-hemolysin+using+a+liposome+display.">In vitro evolution of &#945;-hemolysin using a liposome display.</a> Proc Natl Acad Sci USA. 110 (42), 16796-16801 (2013).</li><li>Whittenton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+asymmetric+liposomal+nanoparticles+for+encapsulation+of+polynucleotides.">Evaluation of asymmetric liposomal nanoparticles for encapsulation of polynucleotides.</a> Langmuir. 24 (16), 8533-8540 (2008).</li><li>Natsume, Y., Toyota, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+vesicles+containing+microspheres+with+high+volume+fraction+prepared+by+water-in-oil+emulsion+centrifugation.">Giant vesicles containing microspheres with high volume fraction prepared by water-in-oil emulsion centrifugation.</a> Chem Lett. 42 (3), 295-297 (2013).</li><li>Pontani, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+an+actin+cortex+inside+a+liposome.">Reconstitution of an actin cortex inside a liposome.</a> Biophys J. 96 (1), 192-198 (2009).</li><li>Nishimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis+inside+giant+unilamellar+vesicles+analyzed+by+flow+cytometry.">Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry.</a> Langmuir. 28 (22), 8426-8432 (2012).</li><li>Fujii, S., Matsuura, T., Sunami, T., Nishikawa, T., Kazuta, Y., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+display+for+in+vitro+selection+and+evolution+of+membrane+proteins.">Liposome display for in vitro selection and evolution of membrane proteins.</a> Nat Protoco. 9 (7), 1578-1591 (2014).</li><li>Hamada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+asymmetric+cell-sized+lipid+vesicles+from+lipid-coated+water-in-oil+microdroplets.">Construction of asymmetric cell-sized lipid vesicles from lipid-coated water-in-oil microdroplets.</a> J Phys Chem B. 112 (47), 14678-14681 (2008).</li><li>Levine, R. M., Pearce, T. R., Adil, M., Kokkoli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+liposome-encapsulated+plasmid+DNA+for+gene+delivery.">Preparation and characterization of liposome-encapsulated plasmid DNA for gene delivery.</a> Langmuir. 29 (29), 9208-9215 (2013).</li><li>Saito, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-resolved+tracking+of+a+minimum+gene+expression+system+reconstituted+in+giant+liposomes.">Time-resolved tracking of a minimum gene expression system reconstituted in giant liposomes.</a> ChemBioChem. 10 (10), 1640-1643 (2009).</li><li>Yanagisawa, M., Iwamoto, M., Kato, A., Yoshikawa, K., Oiki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oriented+reconstitution+of+a+membrane+protein+in+a+giant+unilamellar+vesicle:+experimental+verification+with+the+potassium+channel+KcsA.">Oriented reconstitution of a membrane protein in a giant unilamellar vesicle: experimental verification with the potassium channel KcsA.</a> J Am Chem Soc. 133 (30), 11774-11779 (2011).</li><li>Saito, A. C., Ogura, T., Fujiwara, K., Murata, S., Nomura, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+micrometer-sized+artificial+objects+into+live+cells:+a+method+for+cell-giant+unilamellar+vesicle+electrofusion.">Introducing micrometer-sized artificial objects into live cells: a method for cell-giant unilamellar vesicle electrofusion.</a> PLOS ONE. 9 (9), e106853 (2014).</li><li>Murata, S., Konagaya, A., Kobayashi, S., Saito, H., Hagiya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+robotics:+a+new+paradigm+for+artifacts.">Molecular robotics: a new paradigm for artifacts.</a> New Gener Comput. 31 (1), 27-45 (2013).</li><li>Hagiya, M., Konagaya, A., Kobayashi, S., Saito, H., Murata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+robots+with+sensors+and+intelligence.">Molecular robots with sensors and intelligence.</a> Acc Chem Res. 47 (6), 1681-1690 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The specific gravities of the inner aqueous dispersion medium and the outer aqueous solution must be chosen carefully. For the w/o emulsion to precipitate into the outer aqueous solution during centrifugation, the specific gravity of the inner aqueous dispersion medium must be larger than that of the outer aqueous solution. We tried to prepare GVs using inner and outer solutions without sugars, but we obtained no GVs under these conditions, because the inner aqueous solution did not have enough mass to cross the interference between the two phases. If there is a large osmotic pressure difference between the two solutions, GVs that precipitate into the outer aqueous solution may shrink or rupture. Therefore, the osmotic pressure inside and outside the GVs must be equal. To accomplish this, we used sucrose as a solute in the inner aqueous dispersion and glucose as a solute in the outer aqueous solution; both sugars were at the same concentration. Both salt22 and sugar23,35,38 have been used for such purposes, but sugar is usually employed because it is less toxic and more soluble than salt. However, if too much sugar is added, the GVs may come into contact with the bottom of the cover glass and collapse. There are several strategies for avoiding this. One strategy is to reduce the specific gravity difference between the inner and outer aqueous solutions so that the GVs precipitate under mild conditions; specifically, the supernatant of the precipitated GV dispersion can be exchanged with a solution that is identical to the inner aqueous solution to reduce the possibility of vesicular rupture by adhesion to the cover glass as a result of buoyancy. Another strategy is to precoat both of cover glasses with a thin lipid film29.
Proper preparation and incubation of the emulsion are important. We used liquid paraffin to prepare the oil solution. Mineral oil26,31, dodecane21,33, and squalane33,39 are also often used as solvent oils. Because these materials vary in specific gravity, viscosity, and surface tension, different numbers of vesicles form even when the same centrifugation conditions are used33. To obtain the vesicles with the target properties, it is essential to optimize the specific gravities and viscosities of the inner and outer aqueous solutions as well as the centrifugal acceleration.For preparation of the oil phase, incubation must occur at high temperature and in a dry environment such as an incubator or a dehydrator. In this study, we heated the liquid paraffin to 80 &#176;C to completely dissolve the lipid molecules.In addition, the emulsion should be prepared only as needed and should be immediately subjected to centrifugation because it is unstable just after it is prepared and the w/o droplets readily fuse to one another. The emulsion can be prepared in large quantities by sonication, vortexing, or tapping. However, using a homogenizer allows for rapid preparation of large amounts of emulsion and easier emulsification in oil with a high viscosity. It is also important that the emulsion be layered on the outer aqueous solution gently and rapidly and then chilled at 4 &#176;C. To shorten the time between emulsification and centrifugation, the oil-outer aqueous solution system can be prechilled before the emulsion is layered on it, and the whole system can then be centrifuged immediately.If the white turbidity appears faster or slower than usual, the mechanical homogenizer must be thoroughly rinsed to remove cleaning detergents. In addition, before emulsification, the oil solution, inner aqueous solution, and emulsifier must be returned to room temperature.
Proper centrifugal acceleration is also important. By centrifuging at 18,000 x g, we were able to prepare GVs with a single interior material encapsulated at a high concentration. When the encapsulation of multiple materials is required, it is better to reduce the centrifugal acceleration. For example, an actin assembly system encapsulating seven compounds was achieved with centrifugation at less than 350 x g35. In cases in which centrifugation is undesirable, GVs can be obtained by adjusting the sugar concentration or by precipitating the emulsion under the influence of its own mass38,40,41.
The method reported herein has two major limitations. One is that oil molecules (paraffin, in this case) can be solubilized in the GV membrane, as has been pointed out by the Weitz group21. When insertion of a membrane protein into the GV membrane is desired, the effects of co-existing oil molecules on the protein must be considered. The other limitation is the variation of volume fraction. In this study, we estimated that the volume fractions of microspheres in the GVs ranged from 4-30 vol%; the volume fractions were not identical to the volume fraction of the inner aqueous solution used for GV preparation. Although we were able to encapsulate microspheres in the GVs at a volume fraction high enough for microscopy observation, this method is not suitable for the preparation of GVs with a uniform volume fraction distribution. It has been reported that the distribution of microsphere volume fractions changes during centrifugation34.
The w/o emulsion centrifugation method is commonly used for the formation of GVs containing encapsulated materials. However, few reports have described the preparation of GVs encapsulating microscale materials34,42. Recently, molecular robots containing an encapsulated DNA device or a molecular device have been constructed43,44. GVs with compartments are the first choice for these kinds of applications; therefore, techniques, such as ours, that could be used for encapsulating magnetic microspheres and microspheres with diverse surface functionalization can be expected to be useful44.

The constructive, or synthetic, biology approach is a fascinating avenue for exploring the boundary between living and nonliving matter. Because the cell is the minimum unit required for life as we currently understand it, various researchers have attempted to construct artificial cells from simple, well-understood chemicals so that phenomena that occur within such artificial cells can be studied, with the ultimate goal of elucidating the origins of life and studying the fundamental functions of living cells1,2,3. In particular, vesicles4, which are spherical microcompartments made of amphiphilic molecules and can encapsulate biological molecules such as proteins5,6 and DNA7,8,9,10, have often been used as models of biological membranes.
Vesicles can be classified as small (defined as having a diameter of &#60;100 nm), large (diameter &#60;1 &#181;m), or giant (diameter &#62;1 &#181;m). Giant vesicles (GVs) have been studied extensively because they are similar to living cells in size, shape, and structure. Owing to the size of GVs, morphological changes in GV membranes can easily be observed in real time under an optical microscope.
Several methods for preparing GVs have been reported11, including the hydration method12,13, the freeze-thaw method14, the electroformation method15,16, and the fluidic device method17,18. However, encapsulating proteins and other macromolecules in GVs at high concentrations by means of these methods is difficult. In particular, it is extremely challenging to encapsulate biological materials in sufficient quantity (20-30 vol%) to mimic the crowded environment inside cells19,20. To form GVs instantly, Weitz and coworkers established a water-in-oil (w/o) emulsion centrifugation method21,22. This method has five important features. First, because GVs prepared by this method have low lamellarity23,24, their membranes are so thin that they can be deformed easily. GV membrane deformation induced by FtsZ (a bacterial cell division protein), tubulin, and other macromolecules has been studied25,26,27,28, and we observed polyhedron-like deformation of GV membranes induced by encapsulation of microspheres29,30. Second,membrane proteins can be inserted into the vesicular membrane by this method, albeit with difficulty31. For example, the Yomo group used this method to study the in vitro synthesis and pore-forming activity of the membrane protein &#945;-hemolysin32. Third, it is possible to generate asymmetric GVs in which the lipid components of the inner and outer leaflets are different22. For example, Whittenton et al. generated asymmetric GVs with cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides, and with neutral lipids on the outer leaflet to decrease toxicity and nonspecific cellular uptake33. Fourth, the concentration and volume fraction of substances inside the GVs can be relatively high28,34. Fifth, multiple types of materials can be encapsulated35. For example, Nishimura et al. encapsulated an in vitro transcription-translation system into GVs and used the system to express green fluorescent protein (GFP) within the GVs36. These five features make w/o emulsion centrifugation an indispensable method for generating cell-mimicking GVs.
In previous work, GVs generated by centrifugation were collected by means of a syringe equipped with a long 16 G stainless steel needle containing some of the final aqueous solution22. In the hands of inexperienced technicians, this collection method could easily result in contamination of the GV with some of the oil. In this study, we used the w/o emulsion centrifugation protocol developed by the Yomo group23,37, in which precipitated GVs are collected through a hole opened at the bottom of the centrifuge tube in which they are prepared. We prepared GVs encapsulating 1.0&#160;&#181;m microspheres, which are similar in size to intracellular organelles. The use of microspheres allowed us to estimate their concentration by calculating their volume fraction. Establishment of a method for preparation of GVs in which materials are densely packed is an important step for creating artificial cells. To confirm the utility of our protocol for various types of inner materials, we also demonstrated that GFP and a small water-soluble fluorescent molecule (uranine) could be encapsulated in the GVs.

<table><tbody><tr><td>&lt;strong&gt;REAGENTS:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1,2-Dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375C</td><td>Vesicular membrane molecule</td></tr><tr><td>Texas Red 1,2-dihexadecanoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE)</td><td>Thermo Fisher Scientific</td><td>T1395MP</td><td></td></tr><tr><td>1 M Tris-HCl (pH 7.5)</td><td>Nippon Gene Co.</td><td>318-90225</td><td>Buffer solution</td></tr><tr><td>D(+)-Glucose</td><td>Wako Pure Chemical Industries</td><td>049-31165</td><td>For outer water solution</td></tr><tr><td>Sucrose</td><td>Wako Pure Chemical Industries</td><td>196-00015</td><td>For inner water solution</td></tr><tr><td>Polybead carboxylate microspheres 1.0 &amp;micro;m</td><td>Polysciences</td><td>08226-15</td><td>Nonfluorescent, 2R = 1.0 &amp;micro;m</td></tr><tr><td>Fluoresbrite YG carboxylate microspheres 1.0 &amp;micro;m</td><td>Polysciences</td><td>15702-10</td><td>Fluorescent, 2R = 1.0 &amp;micro;m</td></tr><tr><td>Fluoresbrite YG carboxylate microspheres 0.10 &amp;micro;m</td><td>Polysciences</td><td>16662-10</td><td>Fluorescent, 2R = 0.10 &amp;micro;m</td></tr><tr><td>Fluorescein sodium salt (uranine)</td><td>Sigma Aldrich Japan</td><td>F6377-100G</td><td></td></tr><tr><td>GFP standard (recombinant)</td><td>Vector Laboratories</td><td>MB-0752</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EQUIPMENT:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge 5427R</td><td>Eppendorf&amp;nbsp;</td><td>5409000233</td><td>Centrifuge rotor: FA-45-48-11</td></tr><tr><td>Physcotron</td><td>Microtec Co.</td><td>NS-310EIII</td><td>Handy microhomogenizer</td></tr><tr><td>Generator shaft</td><td>Microtec Co.</td><td>NS-4</td><td>Homogenizer attachment</td></tr><tr><td>Frame-Seal incubation chambers for &lt;em&gt;in situ &lt;/em&gt;PCR and hybridization</td><td>Bio-Rad Laboratories</td><td>SLF0201</td><td>Hybridization chamber volume: 25 &amp;micro;L&lt;br/&gt; Chamber size: 9 &amp;times; 9 &amp;times; 0.3 mm</td></tr><tr><td>Frame-Seal incubation chambers for &lt;em&gt;in situ&lt;/em&gt; PCR and hybridization</td><td>Bio-Rad Laboratories</td><td>SLF0601</td><td>Hybridization chamber volume: 65 &amp;micro;L&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;br/&gt; Chamber size: 15 &amp;times; 15 &amp;times; 0.3 mm</td></tr><tr><td>Microman E</td><td>Gilson</td><td>FD10006</td><td>Pipette for viscous liquid. We measured liquid paraffin by using this.</td></tr><tr><td>Inverted microscope</td><td>Olympus Co.</td><td>IX73</td><td></td></tr><tr><td>Mirror unit</td><td>Olympus Co.</td><td>U-FBNA</td><td>Excitation filter: 470 - 495 nm&lt;br/&gt; Emission filter: 510 - 550 nm</td></tr><tr><td>Mirror unit</td><td>Olympus Co.</td><td>U-FMCHE</td><td>Excitation filter: 565 - 585 nm&lt;br/&gt; Emission filter: 600 - 690 nm</td></tr></tbody></table>

preparation of giant vesicles encapsulating microspheres by centrifugation of a water-in-oil emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

The w/o emulsion centrifugation method is illustrated photographically and schematically in Figure 1. The schematic image in Figure 1&#160;suggests that the most important determinant of the success of this method is that the specific gravity of the inner aqueous solution must be larger than that of the outer aqueous solution, so that the GVs will precipitate during centrifugation. In addition, the formation of a lipid monolayer at the w/o interface requires that the system be chilled for 10 min after the emulsion is layered on the outer aqueous solution. Because the GVs form by transfer of emulsion droplets across the w/o interface, the osmotic pressures in the inner and outer aqueous layers must be the same. As a control experiment, we also prepared GVs containing no microspheres by means of the process shown in Figure 1 and step 1.3, except that the inner aqueous solution was prepared without microspheres.
Collection of the precipitated GVs after centrifugation is shown in Figure 2. In addition to the semitransparent phase at the very bottom of the microtube, we also observed a white, turbid intermediate phase in the outer aqueous solution (Figure 2a). This intermediate phase was rich in aggregations of microspheres and oil, whereas the bottom phase contained the GVs. Therefore, after piercing the bottom of the microtube with a pushpin (Figure 2b), we collected only the first drop (Figure 2c), which contained massive amounts of GVs. It is important to make sure that no more than two drops are collected; any additional drops may contain aggregations of microspheres and lipids, which will result in a lower density of GVs. The obtained vesicular dispersion often contained encapsulated materials outside of the GVs because the GVs often rupture during centrifugation. To obtain only GVs, a sorting method such as dialysis, gel filtration, or fluorescence-activated cell sorting should be chosen, owing to the sizes of the encapsulating materials. If necessary for the purpose for which the precipitated GVs are to be used, they can be diluted with the outer aqueous solution. In some cases, the intermediate phase extended to the bottom of the tube, suggesting that any vesicles that formed were held together by the oil.
We obtained differential interference microscopy and fluorescence microscopy images of the GVs without microspheres (Figure 3a, 3b) and with microspheres (Figure 3c-3f). Consistent with previous reports23,37, GVs with low lamellarity were formed by our protocol. Lipids conjugated with Texas Red DHPE, which emits red fluorescence, were used so that vesicle formation could be directly confirmed by visualization of the thin membrane. Of the 160 GVs that we obtained, 55 encapsulated microspheres and 105 were empty, giving a ratio of encapsulation of 34%.
We determined the volume fraction (&#966;, vol %) of microspheres in the GVs by means of the following method. Because each GV contains dozens to several hundred microspheres, counting all the microspheres under an optical microscope is challenging. Therefore, we mixed the nonfluorescent 1.0 &#181;m microspheres with a small amount of fluorescent microspheres, which were manually counted under the fluorescence microscope. The total number (N) of encapsulated microspheres was calculated by multiplying the number (n) of manually counted fluorescent microspheres by 20 (based on the original 95:5 [v/v] ratio of nonfluorescent to fluorescent microspheres). The value of &#966; was then estimated as Nv100/V, where v is the volume of the microspheres and V is the volume of the individual GV. Note that estimation of N from n gives rise to counting errors, and these errors must be taken into account when calculating &#966;. The value of &#966; was estimated from N, which was directly calculated as 20n, and this in turn resulted in the probability that &#966; accurately represents the true value is &#60;50%. In fact, N fluctuates to some extent around 20n, so we need to consider it as 20(n &#177;i), where i is the error in n. We estimated i in order that a probability of n &#177;i could be more than 50% obtained on the basis of a Poisson distribution. We estimated i, which in turn allowed us to calculate 20(n &#177; i) and values of &#966; that included counting errors for GVs with diameters of 10 and 15 &#181;m (Table 1). According to this procedure, the volume fraction of microspheres in the GV shown in Figure 3c was estimated to be 11 &#177;3 vol %. The precision of the calculated volume fraction was 10-30%.
Our results indicate that we successfully encapsulated 1&#160;&#956;m microspheres in GVs at a high volume fraction. We were also able to encapsulate other materials into 100 mol % DOPC GVs using the same outer aqueous solution and the same protocol (Figure 4). Specifically, GVs containing 0.1&#160;&#181;m microspheres were prepared from a Tris-buffered solution containing 0.1 M Tris-HCl (pH 7.5), 0.15 M sucrose, and 0.5 vol % fluorescent microspheres by means of the protocol described for GVs containing 1.0-&#181;m microspheres (Figure 4a). Following the protocol described above, DOPC GVs containing GFP (0.1 M Tris-HCl [pH 7.5], 0.15 M sucrose, and 100 &#181;g/mL GFP; Figure 4b) and GVs containing uranine (0.1 M Tris-HCl [pH 7.5], 0.15 M sucrose, and 30 &#181;M uranine; Figure 4c) were also prepared.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>n</td>
			<td>N</td>
			<td>&#966; (10-&#181;m diameter)</td>
			<td>&#966; (15-&#181;m diameter)</td>
		</tr>
		<tr>
			<td>3</td>
			<td>60 &#177; 20</td>
			<td>6.0 &#177; 2.0</td>
			<td>1.8 &#177; 0.6</td>
		</tr>
		<tr>
			<td>4</td>
			<td>80 &#177; 20</td>
			<td>8.0 &#177; 2.0</td>
			<td>2.4 &#177; 0.6</td>
		</tr>
		<tr>
			<td>5</td>
			<td>100 &#177; 20</td>
			<td>10 &#177; 2</td>
			<td>3.0 &#177; 0.6</td>
		</tr>
		<tr>
			<td>6</td>
			<td>120 &#177; 30</td>
			<td>12 &#177; 4</td>
			<td>3.6 &#177; 1.2</td>
		</tr>
		<tr>
			<td>10</td>
			<td>200 &#177; 32</td>
			<td>20 &#177; 3</td>
			<td>5.9 &#177; 0.9</td>
		</tr>
		<tr>
			<td>20</td>
			<td>400 &#177; 45</td>
			<td>40 &#177; 5</td>
			<td>12 &#177; 2</td>
		</tr>
		<tr>
			<td>30</td>
			<td>600 &#177; 55</td>
			<td>-</td>
			<td>18 &#177; 2</td>
		</tr>
		<tr>
			<td colspan="4">a Errors were determined as described in the text; n = number of manually counted microspheres; N = total number of encapsulated microspheres.</td>
		</tr>
	</tbody>
</table>
Table 1: Numbers and Volume Fractions (&#966;, vol%) of Microspheresa. a Errors were determined as described in the text; n = number of manually counted microspheres; N = total number of encapsulated microspheres.
<img alt="Figure 1" src="/files/ftp_upload/55282/55282fig1.jpg" /> 
Figure 1: Flow Chart and Schematic Depiction of W/O Emulsion Centrifugation Method. (a) Oil solution consisting of DOPC and Texas Red DHPE (100:0.3 molar ratio) in liquid paraffin. (b) Inner aqueous dispersion consisting of sucrose and microspheres in Tris-HCl buffer. (c) Outer aqueous solution consisting of glucose in Tris-HCl buffer. (d) Mixture of 1 ml of oil solution and 300 &#181;l of inner aqueous dispersion. (e) Emulsification with a homogenizer (10,000 rpm, 2 min). (f) The w/o emulsion. (g) Layering of 300 &#181;l of the emulsion on 1 ml of the outer aqueous solution. (h) Precipitated GVs just after centrifugation. (i) Schematic depiction of the principle of the w/o emulsion centrifugation method. The black arrow indicates the direction of centrifugal acceleration. <a href="http://cloudflare.jove.com/files/ftp_upload/55282/55282fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55282/55282fig2.jpg" /> 
Figure 2: Piercing of the Microtube. (a) Precipitated GVs just after centrifugation (also depicted in Figure 1h). The red ellipse indicates the droplet of the precipitated GV dispersion. (b) Piercing of the microtube with a pushpin. The pellet containing the GVs was obtained by piercing the microtube near the bottom with a pushpin and collecting droplets from the hole. (c) Droplet of the precipitated GVs (indicated by the yellow arrow) dispersion diluted with the outer aqueous solution. <a href="http://cloudflare.jove.com/files/ftp_upload/55282/55282fig2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55282/55282fig3.jpg" /> 
Figure 3: Microscopy Images of GVs. (a)&#160;Differential interference contrast microscopy image of a GV without microspheres. The diameter of GV was about 10 &#181;m. Scale bar = 10 &#181;m. (b) Fluorescence microscopy image of a GV without microspheres. The red fluorescence was emitted by Texas Red DHPE. (c) Differential interference contrast microscopy image of a GV containing microspheres. (d) Fluorescence microscopy image of 1.0&#160;&#181;m microspheres (YG carboxylate microspheres) inside a GV. The number of counted fluorescent microspheres (n) was 6. We estimated the errors (i = 2) based on Poisson distribution. Then we estimated total number of inner microspheres (N = 20 (n &#177;i)) was 120 &#177;40. It was calculated that the GV contained 1.0&#160;&#181;m microspheres at a volume fraction (&#966; = Nv100/V) of approximately 11 &#177; 3 vol%, where v is the volume of the microspheres and V is the volume of the GVs. (e) Fluorescence microscopy image of a GV membrane. The red fluorescence was emitted by Texas Red DHPE. (f) Merged image of the images in panels d and e. <a href="http://cloudflare.jove.com/files/ftp_upload/55282/55282fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55282/55282fig4.jpg" /> 
Figure 4: Differential Interference Contrast and Fluorescence Microscopy Images of GVs with Different Encapsulated Materials. Differential interference contrast microscopy (top) and fluorescence microscopy (bottom) images of GVs containing (a) 0.1&#160;&#181;m microspheres, (b) GFP, and (c) uranine. Scale bars = 10 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55282/55282fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion at JoVE.com

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

preparation-giant-vesicles-encapsulating-microspheres-centrifugation

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JoVE Journal

Biochemistry

14.4K Views.  Japan Women's University. Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials. 

Video: Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Bioengineering

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Chemistry

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

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