Investigating cellular electroporation using planar membrane models and miniaturized devices

This thesis focuses on increasing our understanding of the electroporation process. Electroporation is a technique employed to introduce foreign molecules into cells that can normally not pass the cell membrane. By applying a short but high electric field, pores appear in the membrane through which these molecules can enter the cell. This process is crucial for a number of biotechnological and medical applications (such as drug delivery, particle delivery and gene transfection). This technique is studied with model systems for the cell membrane (bilayer lipid membranes or BLMs) as well as in cells. The membrane models are here employed to study the effect of the membrane composition on the pore formation process. The composition is altered by the addition of different types of phospholipids, cholesterol and proteins and the complexity of the models is increased from binary via ternary to quaternary systems. The results show that membrane composition can have a large effect on the potential required to form pores in the membranes. In addition to this, a microfluidic system for BLM experimentation is developed. In this device, the BLMs are created vertically, allowing for combining electrical and optical measurements. This is a major advantage over a conventional system where only electrical analysis is feasible. The main purpose of this device is as a platform for protein studies for drug screening purposes. Lastly, a cell monolayer electroporation device is employed to verify the hypothesis from the previous results with BLMs that polarized cells become electroporated at a different applied potential than non-polarized cells. In this device, cells are grown onto a layer of hydrogel on top of an electrode substrate or onto a bare electrode substrate, to induce membrane polarization or not

Towards simultaneous electrical and optical investigation of BLMS using a novel microfluidic device

We firstly describe the influence of the phospholipid (PL) composition of bilayer lipid membrane on their electrical properties: (i) the more unsaturations in the tail, the earlier the BLM breakdown and (ii) the bulkier the head group, the less stable the membrane. Secondly, we design and fabricate novel devices that couple such electri-cal characterization to optical investigation and that enable the preparation of asym-metrical membranes: a “macro” device including a drilled PMMA plate as well as microfluidic device consisting of a glass-teflon foil-glass sandwich

Device for measuring the ammonia content in a gas mixture

The invention relates to a device for measuring the ammonia content in a gas mixture. The device comprises at least one inlet and one outlet for the gas mixture, between which is situated an ammonia measuring system connected thereto. The device also comprises first regulating means for regulating the gas mixture flow through the inlet, and second means which are adapted such that they allow a measurement of the ammonia content which is substantially independent of the gas mixture flow rate. The device is preferably applied for measuring the ammonia content in exhaled air

Electroporation in Microfluidic devices

Crossing the plasma cellular membrane for loading of exogenous substances or accessing the intracellular medium is essential for cell engineering and transfection, cell analysis, or controlled extraction of the cellular content. Various chemical and physical techniques have been developed to open up the cell membrane and allow molecular exchange between the extra- and intracellular environments. Electroporation, which relies on the use of a high external electric field to permeabilize the cell membrane, is the most popular physical technique: not only it avoids the use of viral material, but the cell transfection yield is also enhanced compared to chemical approaches. However, while electroporation is currently used on a daily basis for the transformation of a great variety of cells, it still suffers from a low success rate when it is performed in bulk in a cuvette, at the level of an mL-sized cell population. Furthermore, the use of high voltages in the kV range as required in such cuvettes gives rise to various issues, such as Joule heating, creation of bubbles through electrolysis of water, and generation of reactive species, which all compromise the success of the electroporation treatment. Using miniaturized and/or microfluidic devices helps solving these issues while enhancing the overall electroporation success rate, by bringing enhanced control on the process and requiring voltages as low as a few volts. In this chapter, after a short introduction to microfluidics, the unique features this technology can offer for cellular electroporation are discussed. Next, different classes of microfluidic devices for cell electroporation are presented, which are suitable for the treatment of individual cells or small cell populations. Finally, promising applications of microscale cellular electroporation are discussed

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V-th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V-th similar to 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV <= V-th <= 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V-th similar to 250 mV) followed by a noticeable destabilization (V-th similar to 100 mV at 20%). Interestingly, the presence of a model protein, alpha-hemolysin, dramatically disfavours membrane poration and V-th shows a 4-fold increase (similar to 800 mV) from a protein density in the membrane of 24 x 10(-3) proteins/mu m(2). In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation. (C) 2009 Elsevier B.V. All rights reserved

Determination of the electroporation onset of bilayer lipid membranes as a novel approach to establish ternary phase diagrams: example of the l-α-PC/SM/cholesterol system

The lipid matrix of cell membranes contains phospholipids belonging to two main classes, glycero- and sphingolipids, as well as cholesterol. This matrix can exist in different phases, liquid disordered (l(d)), liquid ordered (l(o)) and possibly solid (s(o)), or even a combination of these. The precise phase composition of a membrane depends on its molecular content and more specifically on the presence and amount of cholesterol. This in turn dictates the membrane properties. In this work, the resistance of membranes to the process of electroporation is studied and related to the membrane phase composition. Specifically, the threshold voltage for electroporation is measured (V-th) when DC pulses with increasing amplitude are applied to membranes prepared from various mixtures of a glycerolipid (Heart PC (L-alpha-PC)), a sphingolipid (Egg SM (SM)) and cholesterol (Ch), introduced in various ratios. Binary mixtures (L-alpha-PC/Ch, L-alpha-PC/SM, SM/Ch) and L-alpha-PC/SM/Ch ternary mixtures are successively employed. For all binary and ternary systems, dramatic changes in V-th are measured as a function of the membrane molecular composition, and the variation patterns of V-th are successfully correlated with the membrane phase composition. Interestingly, the measure of the electroporation onset can be employed as a novel methodology to establish ternary phase diagrams, and this is illustrated with the L-alpha-PC/SM/cholesterol ternary system