GB2523579A - Modular serial multi-chamber flow-through electroporation device - Google Patents

Abstract

A flow-through electroporation device comprises at least two electrodes arranged in series and separated by at least one insulator in a flow direction. Electrodes and insulators are preferably fitted together by a thread. The device may comprise a plurality of co-linear chambers arranged in series. A plurality of meshes is included to provide contact between adjacent electrodes and insulators. One or more mesh distancers may play a role in electric field homogenisation. The device may be used in food processing applications.

Description

Title: Modular Serial Multi-Chamber Flow-Through Electroporation Device

Field of Invention

The invention is related to the electroporation systems for continuous irreversible or reversible poration of cells that can be used for laboratory scale operation.

Background of Invention

Electroporation is a procedure that utilises a series of short high-voltage pulses to elevate permeability of a cell membrane and is used for cell destruction, irreversible electroporation or for introduction/extraction of substances in/from the living cell, reversible electroporation. So far it has found a wide variety of uses in bio-technology. Electroporation in continuous flow is a special case of electroporation that is being used in food processing, inactivation of microorganisms in of water in cases where chemical disinfectants cannot be used as well. Reversible electroporation, when cell are only temporaly affected by electric pulses is used in different applications among which continuous flow is interesting for gene electrotransfer in larger scale or for extraction of different commercially interesting substances from plant cell cultures (M. Konduter, 0. Miklavi4 Electroporatian in biological cell and tissue: on overview, Electrotechnologies for Extra ction from Food Plonts ond Biomateriols (2008) doi: lo.1007/978-0-387-79374-o_1). Electroporation chamber design is one of the most important steps for efficient electroporation. There are several designs reported in the literature mostly designed for food processing (a Gerloch,N. Alleborn, A. Boors, A. Delgodo, I. Moritz, D. Knorr, Numerical simulations of pulsed electricfieldsforfood preservotion: A review, innovative Food Science and Emerging Technologies, (2008), doi:10.1O16/j.ifset.2008.02.001, S. Toepfl, Pulsed electric field food processing -industrial equipment design and commercial applications, Stewort Post harvest Review, (2012), doi:10.2212/spr.2012.2.4). In most cases, homogenous electric fields and laminar flow are desired to obtain equal conditions of poration for all cells in the flux. Most common designs are: planar, co-axial or co-linear. Planar designs produce homogeneous electric fields with two parallel metal plates with flow in-between; however, the gap between the electrodes should be as small as possible to obtain electric fields of sufficient strength at reasonable voltage differences between the electrodes. Sealing of such chambers is also problematic. Co-axial chambers produce non-homogeneous electric fields with two concentrically placed electrodes; the gap between the electrodes must also be kept at minimum. The positioning of the electrodes and efficient flux path is problematic for this type of electrodes. Co-linear electrodes produce non-homogeneous electric fields with two ring electrodes of the same diameter placed on the same axis next to each other; however, the non-homogeneity can be controlled redesigning the geometry of insulators (R. Buckaw, P. Baurnann, S. Schroeder, K. Knaerzer, Effect of dimensions and geometry of co-field and co-linear pulsed electric field treatment chambers on electric field strength and energy ut/I/sat/on, Journal of Food Engineering. (2011), doi:1a1015/j.jfoodeng.2011.03.019) or by using additional meshes attached to the electrodes (H. Jaeger, N. Meneses, D. Knorr, Impact of PEP treatment inhomogeneity such as electric field distribution, flaw characteristics and temperature effects on the inactivation of E. coli and milk alkaline phasphotase, innovative Food Science and Emerging Technologies, (2009), doi:10. 101 6/j.ifset.2009.03.001). Sealing of the electrodes is relatively simple; however, the flux through the chamber must have a large through-section to prevent problems with relatively short time that each cell spends in the area of sufficient electric field strength. The efficiency of the chamber is defined by electric field strength and the number of pulses received by each cell, however, both effects show saturation characteristics and must be in balance to achieve optimal functioning of the system (M. Kanduer, D. MikIavi& Electroporation in biological cell and tissue: an overview, Electrotechnologies for Extraction from Food Plants and Biomaterials (2008) doi: 10.1007/978-0-387-79374-0_i). Transitions from small to large through-sections of flows can in addition cause significant turbulences in the flow and can thus affect the chamber efficiency.

In the presented invention we solved a problem of large through-section flows by combining several co-linear chambers in series. We also solved the problem of non-uniformity of electric field distribution throughout the treatment chamber by nesting the edges of the electrodes into the insulator (mesh distancer), which eliminated the pronounced electric field gradients in the vicinity of the electrode-insulator interfaces. The reduced electric field gradients have positive effect on electric mesh erosion. The removable meshes used in the chambers corrected the electric field homogeneity and improved laminarity of the flow. The modular design allows large number of the chambers to be added to the original construction, thus allowing larger flows through the chamber without efficiency loss.

Summary of the Invention

The invention solves a problem of finding optimal flow/electroporation effect in laboratory conditions. The electroporation device is designed as a modular system that consists of a series of electrodes separated by insulators. Electrodes and insulators are fitted together by a thread. Since the electrodes and insulators are symmetrical, they can form theoretically infinite chain of chambers. At both ends pipeline connectors are added. The larger the number of the chambers, the larger flow can be handled by the electroporation system, assuming each bacterium within the flow must obtain some minimal number of electroporation pulses to achieve certain efficiency of the sterilisation or any other procedure. In large scale operations such modularity does not present significant advantage since each system must be designed to meet pre-set goals. In laboratory environment, however, the conditions of testing may change quite significantly between experiments and precise specifications for electroporation chambers and accompanying equipment cannot be set. Therefore, modularity of the invented device presents significant advantage over custom made flow-through electroporation chambers. The device assembly can be adapted with respect to the flows that are limited by the available pumps) available repeating frequencies of the electroporator, and electroporation properties of the bacteria.

Brief Description of the Drawings

Figure 1: Schematic representation of all types of device modules (electrode insulator and mesh distancer, mesh, 0-ring sealing, electrode, and pipeline connector), and assembled device with two chambers.

Figure 2: Numerical calculation of electric field distribution throughout the treatment chamber -throughout the electrodes, the insulator and liquid volume (single chamber composition)

Detailed Description of the Invention

Laboratory scale flow-through electroporation chambers should operate under large variety of operational conditions. In most cases they are designed to cause irreversible electroporation either to improve extraction of substances from cells in food processing and biopharmaceutics or to inactivate undesired microorganisms in fluids. Each cell must receive sufficient number of high-enough electric field strength pulses to destroy the membrane. As various cell types can be subject to electroporation (microorganisms, fungi, animal, and plant cells, etc.) the conditions for irreversible/reversible electroporation can vary significantly. In order to compensate for the differences, various combinations of settings of pumps and electroporators are not always sufficient. To additionally compensate for the differences and to expand the design space that is covered by electroporation equipment a modular multi-chamber flow-through electroporation device was developed.

The following goals were set: * possibility to use higher flows at relatively low repeating frequency of electroporation pulses, * close-to-homogenous electric field within the chamber, * laminar flow through the chamber to prevent unpredictable motion of the cells through the chamber, * easily expandable modular design, * simple removal of air bubbles from the device, * simple maintenance of the device.

Co-linear devices are most suitable for simple maintenance, except for additional meshes that are required to compensate for significant non-homogeneity of the electric field. To resolve the problem, removable meshes were installed that are thicker at the edges. Thicker edges of the mesh act like spring contacts and ensure sufficient contact with the electrode. The meshes are asymmetrical; flat on one side and with visible edge on the other side. The mesh is placed into the insulator with flat part turned towards its seat within the insulator (see Figure 1) and the electrode is screwed to its final position to ensure a good contact between the mesh and the electrode. The same procedure is applied on the other side of the insulator. The mesh seat in the insulator is designed as a ring that defines the gap between the mashes and thus also the electric field shape and strength. The shape of the mesh distancer plays a key role in electric field homogenisation. The mesh distancer (Figure 1) is of rectangular shape when observed from the axial cross-section such that the mesh remains in close contact with the insulator at the front edges. Because of such design, the non-homogeneities and high gradients of the electric field strength at the edge of electrode-insulator interface of the planar capacitor that is formed with the two meshes remain entirely within the insulator and do not come in contact with the porated fluid (see Figure 2). The threads fixing the electrodes into the insulator are sealed with the 0-ring seal placed in the groove of the electrode (see Figure 1). With the described design the following is achieved: * simple maintenance (the device can be easily disassembled for thorough cleaning if impurities in the fluid get attached to the mesh and in case damage the mesh can be easily replaced), * meshes produce almost homogenous electric field in the chamber, * the number of chambers is simply expandable by adding additional such electrode-insulator objects to the assembly, * the expanded assembly can convey larger fluxes while assuring the required number of electroporation pulses per cell.

To achieve higher fluxes at relatively low pulse repetition frequencies the chambers can be added to the assembly in series. The result of such composition is relatively long device where laminar flows are easier to achieve than in a short device, since transition from small-diameter tubes to the large diameter chamber and back to the tubes cause additional instability of the flow. The effect of transition between the diameters is less significant if the device is longer and the flow can settle back in laminar form before leaving the device. Smaller difference between tubes and chamber diameter also reduces significance of the transient effect. Electrode meshes further reduce the possibility of the turbulent flow through the chamber. The shape of the tube connectors (see Figure 1) is also designed to reduce the possibility of turbulence formation within the chamber. Thus the design with several consecutive chambers is more efficient than the design with single large diameter chamber also in terms of flow laminarity.

Air bubbles may cause severe disturbances to electric field distribution and can interfere with laminar flow. In order to remove air bubbles from the device) it must be mounted in a vertical flow position. The shape of the tube connectors also prevents collection of the air bubbles within the device.