Part:BBa_K260018

Methdod to mass produce vesicles of uniform shape and volume

Vesicle characterization

• Content of the vesicles can be chosen freely. Environment inside vesicles is aquious.
• Vesicle shape: spherical
• Vesicle diameter: 15 µm
• Vesicle volume: ~12 pl
• Vesicle population has a very narrow distribution in size.
• Production efficiency: 5-15 vesicles per second (Fig. 1)
• Vesicle stability: not fully characterized, depends on surfactant used, at least 2 hours; fusion of vesicles observed after >24 hours
• Optical properties: transparent in brightfield microscopy; for observation of fluorescence the surfactant used should be non-fluorescent
• Vesicle isolation through output hole. Vesicle content is released when vesicles transfered to aqueous
• Vesicle conveniently observable in a grid (Fig. 2)

Fig. 1: Vesicle formation in a V-chamber

Fig 2: Different geometries of the intersection between aqueous and oil phase in flow chambers

T shaped junction

Protocol

You will need a silicone waver with the engraved microfluidic system, and a micropump system

Setup of the microfluidic system

The microfluidic system consists of a flow chamber made of Polydimethylsiloxane (PDMS) and a pump system that controls the flow rates of the various liquids into the chamber. Droplets are created within a defined space in the chamber and are propagated along a grid that allows containment and imaging. Two types of chambers have been used, differing in the geometry of the space where droplets were produced. One featured T-junction, and the other a V-junction (Fig. 3).

Fig 3: Different geometries of the intersection between aqueous and oil phase in flow chambers

T shaped junction

V shaped junction

Production of flow chambers

• mix PDMS and curing agent in 10:1 ratio
• degas and pour on wafer with etched microstructures
• polymerize on heat plate at 150 ºC for 30 min
• add unpolymerized PDMS mixture to points on microstructure where microtube inlets are to be pierced
• polymerize on heat plate at 150 ºC for 30 minutes
• remove polymerized PDMS from wafer, cut to fit onto glass cover slide (24 x 60 mm), and use clean needles (0.8 mm) or laser cutter (Trotec Speedy 100TM) to pierce tube inlets
• ionize PDMS and glass slide in plasma chamber for 30 sec to make it reactive
• align PDMS on glass slide and seal
• seal irreversibly by heating on plate at 60ºC for 6 hours

The pumping system (ceDOSYS SP-4) allows control of syringes filled with aqueous material and mineral oil treated with surfactant(1% Span 80), respectively. The syringes access the chamber via the tubing inlets. Two inlets are used to pump in material in the aqueous phase; the remaining one is used for the oil phase. The flow rates of the syringes are controlled via a ceDOSYS user interface software.

Control via pump system:

• two syringes are loaded with 1ml each of material in the aqueous phase; during the first trial, distilled water * another is filled with a 1ml solution of 0.5% span 80 in oil
• use flow rate on ceDOSYS interface to flood the chamber first with oil phase
• gradually introduce aqueous phase and modify rates of both phases until the shear stress breaks the aqueous phase into droplets at the T- or V- junctions in the respective chambers.