Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-03-25
2003-11-25
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S470000, C435S471000, C435S285200
Reexamination Certificate
active
06653136
ABSTRACT:
This invention relates to an apparatus for and method of introducing a substance into an object.
More particularly, but not exclusively, the invention is capable of introducing substances into small objects, such as for example, cellular material or cells. The substance introduced may be a transfecting agent such as: a chemical, molecule, protein, virus, prion or DNA material.
Previously material has been introduced into cells by way of a syringe-like device. These syringe-like devices have to be operated by hand or by complex robotic systems, and in consequence the injection process has been very slow.
The present invention arose to overcome this and other problems associated with syringe-like devices.
It is an object of the invention to increase the efficiency of injection of material into small objects such as cells by the presently-known process of electroporation. In this process cells suspended in a medium are exposed to an electric field sufficiently high to cause the membrane to become permeable. In the present art, cells are suspended in a medium containing the species required to be injected. When the membrane has been made permeable, the species to be injected will either diffuse or be driven by electrophoresis into the cell. After a time the cell membrane will recover from the effects of the pulse and become non-permneable once more. In the present art high concentrations of cells are used, together with macroscopic (ie, with typical minimum dimension of order 100 &mgr;m) electrodes. This means that in general, more than one cell will be in series with each of the field lines in the system—the precise number being random. Hence the field experienced by the cells in the system can very greatly, from below that required for membrane opening, to above that which the cells can stand and remain viable. It is an object of the invention to provide a microfabricated system, with characteristic dimensions considerably smaller than those encountered in presently used electroporation devices, where cells can be electroporated and material injected in conditions which are very similar from cell to cell, allowing uncertainties of field strength and timing to be reduced.
Microfabricated devices for handling, cells and exposing them to electric fields are known. Ayliffe et al (IEEE J MEMS 8(1)50-57 (1999)) show microfabricated channels with micro-electrodes which can be used for electrical impedance measurements on liquids and cells, the latter aimed at detecting the type of cell present and measuring (if possible) some of its properties. The device is not designed for the purpose of electroporation and no mention is made of this. Tanaka et al (U.S. Pat. No. 4,894,343) disclose a microfabricated device for handling cells In wells, designed for the purpose of fusing together two cells located in the same well. Their device comprises an array of wells etched in silicon which are designed to trap the cells, while allowing liquid to move past them through outlets in the bottom of the well. However, the device is not designed to be optimal for electroporation, the present invention uses an improved design.
According to the present invention there is provided an apparatus for introducing a substance into an object comprising: means for introducing the substance to the object; and means for causing permeability of the wall of the object so as to permit said substance to enter said object; characterised in that the means for causing a permeability includes at least one electrode, dimensioned and arranged to form the permeability in the wall of the object upon application of a voltage pulse.
Preferably a characteristic dimension of a channel through, or along, which an object passes or flows is of the order of 50 &mgr;m, more preferably it is less than 30 &mgr;m and most preferably less than 4 times the diameter of the object.
Objects or cells suspended in a liquid medium are introduced into a chamber in which the apparatus(es) is/are located by way of a pump or gravity feed or other suitable fluid displacement mechanism, for example by electro-osmosis.
Preferably at least two electrodes are provided so that the object is located with respect to the electrodes so that a potential difference may be applied in order to render the object wall temporarily permeable.
Means can be provided to restrain or locate the object so that it is positioned with respect to the or each electrode. An advantage of locating the object is that it is positioned in a particular part of a predetermined electric field. Consequently the electric field may be applied with greater precision.
A proximity detector is advantageously included, so that when an object is in the correct location to be in a predetermined part of the electric field, the voltage pulse is applied automatically. Processing means, including electronic logic, may be used to improve and enhance this process.
Preferably there is provided a plurality of the aforementioned apparatuses arranged in an array. An advantage of such an array is that many objects may be acted upon in parallel. This increases throughput.
An array of apparatuses may be formed on a semiconductor substrate, such as for example, silicon or germanium. Proximity detectors, electrodes and processing means may be included on the substrate, for example, in a different layer of an integrated semiconductor structure.
In a particularly preferred embodiment DNA is introduced into living cells by rendering permeable the cell wall by the process of electroporation. DNA then enters the cell from the surrounding medium. Cells are supported in a fluid which is under sufficient pressure to cause cells to move with respect to the electrodes. Means for locating each object with respect to an electrode may comprise a mechanical or electrical structure. An example may be a well or well-like structure, formed for example by back etching a silicon substrate in which the cell locates. A mesh or sieve-like arrangement can be placed at the exit of the well so as to permit passage of fluid but prevent the cell from leaving the well. Preferably a pressure differential established across the substrate urges cells into the well-like structures.
As more cells are located in wells the pressure differential increases because less wells are available, through which fluid may flow. This increased pressure tends to force cells into the wells as they deform relatively easily. One way of preventing this from occurring is to obtain an indication of wells which are occupied and use this information to reduce or increase the pressure differential. This information is readily obtainable as the presence of a cell is known from proximity detectors and a counter in a microprocessor may be used to increment each time a well becomes occupied.
Preferably the apparatus is microfabricated from a biocompatible material. The microfabricated apparatus may include one or more microfabricated channels. These may be formed for example by etching in silicon. Wells or sites for introduction of material may be at a locus in a fluid flow channel.
The channel is preferably narrow, for example, between 1 and 5 times the diameter of the cells (which may typically be around 5-20 &mgr;m) to be electroporated. Such narrow channels are advantageous in electroporation, as a greater proportion of the electroporation voltage may be applied across the cell per se, rather than across the cell, any neighbouring cells and the supporting fluid. This enables the field experienced by the cell to be controlled precisely. In an alternative embodiment the channel may even be narrower than the diameter of the cell in its relaxed state. In this embodiment cells deform and flow along the channel and are in closer contact with the walls.
Alternatively the channel or well may be relatively wide except for a constriction in the region at which introduction of material occurs, the constriction, and/or eletroporation electrodes may be designed so that pores, opened in the cell membrane to allow introduction of material, are preferentially oriented at a source of
Corless Anthony R
Dodgson John
Astrazeneca AB
Ketter James
LandOfFree
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