Bottom%Up(Synthetic(Biology:( Double(Emulsion(and(the(

Transcription

1 Bottom%Up(Synthetic(Biology:( Double(Emulsion(and(the( Artificial(Cell( Christopher W. Coyne a a University of Michigan, Department of Mechanical Engineering Abstract Bottom-up synthetic biology embodies an interdisciplinary approach towards systematically modeling and constructing living systems from biological components, thus achieving functionally indistinguishable products or systems with unique new properties. This emerging field engages engineers, chemists, biologists, and physicists alike, and aims to understand, characterize, and ultimately recreate animate nature. 1 Due to the inherent complexity and redundancy of living systems, bottom-up synthetic biology traditionally begins at the cellular or sub-cellular level. 2 In a synthetic cellular environment, giant unilamellar vesicles (GUVs) can serve as a model platform due to their cell-like membrane structure and size. This project uses double emulsion template, a microfluidic technique for high-throughput vesicle generation, to create lipid bilayer vesicles as model systems that will enable testing and validation of different features of our artificial platelet design. INTRODUCTION As our understanding of living organisms on the larger scale grows more comprehensive, so too does our understanding on the micro-scale. A natural extension of this understanding is the desire to recreate nature, not only to develop novel systems with unique and pertinent applications, but also to unearth a new, deeper knowledge of biology. Bottom-up synthetic biology is a blend of several scientific disciplines that aims to supply this understanding by systematically constructing living systems from synthetic components cell by cell and module by module. This provides a powerful and flexible approach towards developing a functionally indistinguishable, artificial cell with predetermined properties and limitless applications (artificial platelets, for instance). Giant unilamellar vesicles (GUVs), small bubble-like structures with single lipid bilayer membranes, can serve as ideal model platforms for synthetic cells due to their cell membrane-like qualities and easily observable size using optical microscopy. The lipid bilayer chemistry can be tuned to mimic a natural cell membrane, and the vesicle can be constructed to encapsulate material just as a natural cell does (DNA, organelles, etc.). A variety of methods currently exist for producing lipid bilayer vesicles, including microfluidic jetting 3, extrusion 4, and electroformation 5. However, these methods suffer from low throughput, polydispersity, or multilamellarity. Double emulsion template, a technique for mixing normally immiscible liquids in a controlled manner in order to generate lipid bilayer vesicles, offers a robust method for stable, high-throughput, monodisperse, unilamellar vesicle production. In addition to artificial cells,.

2 2 these vesicles are of interest in such industries as cosmetics, food, and drug delivery where a highly regulated release of chemical substances is applicable. The goal of this project was to construct and optimize the double emulsion system in the Allen Liu lab for application in the artificial platelet project. The vesicles produced via double emulsion could subsequently be studied via micropipette aspiration (a method for applying suction to an individual cell in order to understand the structural properties of its membrane), flowed through microfluidic devices (for testing behavior under flow-induced shear stress), and subjected to various other experiments. METHOD Micropipette Fabrication Injection and Collection Tubes The injection and collection tubes (Fig. 1) were fabricated from aluminosilicate cylindrical glass capillary tubes, with 1.00 mm outer diameter and 0.64 mm inner diameter (Sutter Instruments A ). First, capillary tubes were pulled using a Sutter Instruments P-97 Micropipette Puller. To generate the tapered tip conducive to double emulsion template, settings of Pull = 0, Pressure = 500, Time = 200, Velocity = 14, and Heat = [Ramp value + 15] were used. The tips of the injection and collection tubes were then sanded to an outer diameter of 120 µm and 150 µm, respectively. Figure 1. Double emulsion template. inner phase tube injection tube Inner phase tube collection tube viewing chamber The inner phase tube (Fig. 1) was also fabricated from a 1.00 mm outer diameter / 0.64 mm inner diameter aluminosilicate cylindrical glass capillary tube (Sutter Instruments A ). However, the inner phase tube was pulled by hand over a flame. While holding onto either end, the middle of the capillary tube was placed in the flame until it became soft. Then the ends were extended and the capillary tube was removed from the flame, simultaneously. This resulted in a gradual taper with nearly parallel walls. At this point, the extended capillary tube was broken at its narrowest point, resulting in two pipettes that could be processed into inner phase tubes. The pipettes were viewed under a microscope to identify the point at which their outer diameter was 200 µm (the optimal outer diameter for the inner phase tube). The pipettes were scored and broken at this point with a ceramic tile (Sutter Instruments CTS). Outlet The outlet was used to facilitate vesicle collection and to allow the double emulsion device to connect to other microfluidic devices. A ~9 cm section of 1.00 mm outer diameter / 0.64 mm inner diameter aluminosilicate cylindrical glass capillary tube (Sutter Instruments A ) was cut by scoring and breaking. To produce the bend, the middle of the pipette was placed over an open flame (while supporting only one end of the pipette), and the end of the pipette was allowed to fall under gravity until the desired angle was achieved. The inner surfaces of all micropipettes were coated with silane to render them hydrophobic. Double Emulsion Device Construction The double emulsion device used a combination of square and cylindrical glass capillary tubes precisely mounted on glass slides. First, a length of square capillary tube, known as the viewing chamber, was cut to length (Table 1). The viewing chamber was bonded to a glass slide with epoxy at

3 3 3 each end, providing a secure means of attachment without obscuring the view of vesicle production. Table 1. Glass capillary component lengths. Component Glass capillary shape Length (cm) Viewing chamber Square ~2 Injection tube Cylindrical ~3 Collection tube Cylindrical ~3 Injection supporter Square ~0.5 Collection connector Square ~1 Inner phase tube Cylindrical ~2 The injection and collection tubes were then cut to length (Table 1) and gently aligned within the viewing chamber. Since device performance was contingent on proper alignment, the injection and collection tubes were precisely aligned under a microscope. To avoid creep due to residual stresses or imbedded bending moments, a supporting piece, the injection supporter, was cut from a square capillary tube (Table 1) and placed at the end of the injection tube. A similar supporting piece, known as the collection connector, was cut (Table 1) and placed at the end of the collection tube. The collection connector served the additional purpose of connecting the collection tube to the device outlet so that vesicles could be collected. The injection tube was then bonded in place with epoxy, and the epoxy was allowed to dry. Once the injection tube was securely attached, the collection tube was realigned (as securing the injection tube often shifted the device) and subsequently bonded in place with epoxy. The separation between the tips of the injection and collection tubes was approximately µm. The inner phase tube was cut to length (Table 1), and a ~5 cm piece of flexible plastic tubing was attached to the newly cut end. The tip of the inner phase tube was then carefully inserted into the injection tube and secured in place with epoxy. While the depth of insertion of the inner phase tube was not critical, concentricity with the injection tube was crucial for proper inner phase delivery. Once the epoxy had fully cured, the flexible plastic tubing was bonded to the glass slide. This additional measure of securing the plastic tubing ensured that the inner phase tube remained concentric with the injection tube when attaching additional components to the inner phase inlet. Throughout the device construction process thus far, additional glass slides were connected to the original slide as necessary with epoxy. Three 20G, 0.25-inch inner diameter / 0.36-inch outer diameter, 1-inch needles (Becton Dickinson BD ) were used to facilitate fluid delivery to the device. Two small channels were cut out of each needle casing to allow space for a capillary tube (Fig. 2). A needle was then placed 1) over the intersection of the inner phase tube and the injection tube, 2) over the intersection of the injection tube and the viewing chamber, and 3) over the intersection of the viewing chamber and collection tube (Fig. 2). Epoxy was mixed for ~5 minutes in order to achieve a high viscosity, and the needles were bonded in place. A thick epoxy was important at this stage, as a fluidic epoxy would flow into the channels in the needles and block fluid entrance into the device. Figure 2. Three 20G needs were used to facilitate fluid delivery to the double emulsion device. middle phase outer phase Experimental Setup water

4 4 The middle phase, consisting of 35 mol% 1,2dioleyl-sn-glycero-3-phosphocholine (DOPC), 35 mol% 1,2 dipalmitoyl sn glycerol 3 phosphocholine (DPPC) and 30 mol% cholesterol6, was first prepared. This lipid mix was dissolved in a mixture of 40 vol% chloroform and 60 vol% hexane, with a total lipid concentration of 10 mm. The outer and inner phases were then prepared according to Table 2. Table 2. Inner phase and outer phase solutions. Inner Phase Lysate Mix 1 GADD34 Mix 2 T7 DNA (MscL-GFP) Sucrose PVA in PCR water C6-TAC Target Concentration Volume (µl) Outer Phase HEPES KCl Glycerol PVA CaCl2 Sucrose To prepare for vesicle production, the double emulsion device was mounted on the microscope stage, the high-speed camera (Phantom MIROEX22048MM) was connected and engaged, and imagecapturing software (Phantom PCC 2.1) was configured. A plastic syringe was filled with deionized water and connected to the device (Fig. 2) via flexible plastic tubing, and each inlet to the device was subsequently flushed with water to remove any air. During double emulsion production, this water channel provided a means for removing any undesired particles or trapped air from the double emulsion generation interface. The outer, inner, and middle phase solutions were then loaded in syringes. To prevent mixing of the aqueous inner phase within the syringe, the following solutions were drawn up sequentially: 50 µl water, 50 µl hexane, 25 µl inner phase, and finally 25 µl hexane. All syringes were mounted on automated syringe pumps and connected to the double emulsion device via flexible plastic tubing according to Fig. 2. Each solution was carefully connected to avoid trapping any air. The middle phase and outer phase were then run at 350 µl/hr and 3000 µl/hr, respectively. Once single emulsion (oil-in-water droplets) occurred, the inner phase was run at 350 µl/hr. Double emulsion vesicles were then collected at the device outlet. Vesicles were collected in a solution (Table 3) with identical osmolarity to the inner phase to prevent osmotic shock. Table 3. Vesicle collection solution. Target Concentration Collection Solution HEPES KCl Glycerol PVA Glucose DI Water N/A Volume (µl) The final device is shown in Fig. 3. Figure 3. The final double emulsion device, including an inlet for the inner phase (left), middle phase, outer phase, and water (three needles), and an outlet for vesicle collection (right). RESULTS Double emulsion template provided a stable method for high-throughput vesicle generation. Vesicles produced showed high uniformity in size

5 5 3 and shape (Fig. 4), and could be studied via microfluidic manipulation or micropipette aspiration. Figure 4. Double emulsions of size ~40 µm are produced from the injection tube (left) and collected in the collection tube (right). Although very stable, solution dispensation resulted in a limitation in vesicle throughput. Due to the geometry of the device, the injection of the inner phase was discontinuous, which resulted in intermittent production of double emulsion vesicles. While this did not affect the stability of production, it did mitigate the double emulsion yield. With a middle phase flow rate of 350 µl/hr, inner phase flow rate of 300 µl/hr, and outer phase flow rate of 3000 µl/hr, double emulsion production peaked at ~60% of throughput (the remainder consisting of single emulsions). However, this discretization was somewhat beneficial in that it allowed for an inherent reset in the system, which was particularly useful when the inner phase mixed with the outer phase and negated double emulsion production. Additionally, as vesicle production removed lipid molecules from the double emulsion interface, the lipid bilayer was subject to rupture under tensile forces. However, by increasing the middle phase flow rate slightly higher than the inner phase flow rate, the bilayer would remain saturated. With a middle phase flow rate of 350 µl/hr and inner phase flow rate of 300 µl/hr, double emulsion production peaked at ~65% of throughput. Increasing the middle phase flow rate also increased the ratio of (inner phase and middle phase) to (outer phase), which meant less shear stress acted on each double emulsion and larger double emulsions were produced. Due to the mitigated shear stress during production, these double emulsions were less likely to burst during formation. DISCUSSION and CONCLUSIONS The results of this work provide a characterizable method for producing unilamellar, lipid bilayer vesicles, the first building block for artificial platelets. Customizability at this stage, particularly in the artificial platelet context, is a powerful tool. For instance, to accomplish activation by flow, an essential component of platelet functionality, reconstituted mechanosensitive channel of large conductance (MscL) can be incorporated into the lipid bilayer to facilitate calcium influx under flowinduced shear stress. While 40 vol% chloroform and 60 vol% hexane were used in the inner phase solution, 36 vol% chloroform and 64 vol% hexane are recommended by Arriaga et al. 6 We found the 40%:60% solution to result in more stable vesicle lifetimes, perhaps due to the encapsulation of CFE lysate. An investigation of vesicle yield when encapsulating CFE lysate, perhaps in combination with an analysis of the dewetting nature 7, is the subject of future work. Another next step in this work entails investigation of membrane tension, and how membrane tension can be used to trigger biochemical reactions within the vesicle. MscL, which opens under instances of membrane tension, is one way of achieving this behavior. REFERENCES 1. Boldt, Joachim. Synthetic Biology: Origin, Scope, and Ethics. Minding Nature 3,1 (2010). 2. Schwille, Petra. Bottom-Up Synthetic Biology: Engineering in a Tinkerer s World. Science 333, 6047 (2010): Coyne, C.W. et al. Lipid bilayer vesicle generation using microfluidic jetting. Journal of Visualized Experiments 84 (2014). 4. Frisken, B.J. et al. Studies of Vesicle Extrusion. Langmuir 16, 3 (2000).

6 6 5. Méléard, Philippe et al. Giant Unilamellar Vesicle Electroformation: From Lipid Mixtures to Native Membranes Under Physiological Conditions. Methods in Enzymology. Elsevier 465 (2009): Arriaga, Laura R. et al. Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation. Small 10, 5 (2013): Shum, Ho Cheung et al. Dewetting-Induced Membrane Formation by Adhesion of Amphiphile- Laden Interfaces. Journal of American Chemical Society 133 (2011):