Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2002-12-06
2004-03-30
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S285200
Reexamination Certificate
active
06713292
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to electro-fusion and electroporation systems, and more particularly to devices that allow for electro-fusion experiments to be conducted using electroporation systems and devices.
It is well known to fuse cells using a membrane suspended between parallel electrodes. U.S. Pat. No. 6,221,665, which is hereby incorporated by reference for all purposes, discloses one such system for performing cell-cell electrofusion. In the disclosed system, a gentle vacuum is drawn from below a membrane which causes the cells to be sucked-down into the membrane. As shown in
FIG. 1
, a vacuum is created in the region
10
below the membrane
20
using, e.g., a syringe
30
or other vacuum generating device. A potential is then applied to the electrodes, and the membranes of adjacent cells are disrupted. The cell machinery reconstitutes the adjacent membrane such that they become contiguous. This causes fusion but without need for the AC currents to cause dielectrophoresis (movement of the cells in a special electric field such that the membranes of the cells are adjacent) as is the usual method.
It would be advantageous to implement such a fusion system in an electroporation system, for example, to use the DC source of the electroporation system. However, such a fusion system as described above would not be useful in a typical electroporation cuvette system arrangement as the area in which the vacuum port is located is in the same area of electrical contact to the cuvette electrodes. Hence, it would be desirable to provide a simple and efficient means of creating a fusion cuvette from a standard electroporation cuvette without interfering with the electrode contact area.
BRIEF SUMMARY OF THE INVENTION
The present invention provides cuvette inserts adapted and configured to fit within an electroporation cuvette. The inserts each include a support structure that holds a porous membrane. When positioned within the cuvette, the membrane is positioned proximal the cuvette electrodes to facilitate membrane-based fusion of cells. In certain aspects, a tube extends through the support structure to allow for application of negative pressure in a convenient location away from electrode contacts and other components of the cuvette or cuvette holder.
According to an aspect of the invention, an electroporation cuvette insert for creating a membrane-based fusion cuvette from an electroporation cuvette is provided. The insert typically includes a porous membrane having an upper surface and a lower surface, a support structure for holding the membrane, the support structure being of a sufficient dimension to securely match, or fit within, the inner walls of an electroporation cuvette, wherein the support structure creates a substantially sealed chamber within the cuvette below the lower surface of the membrane when inserted into the cuvette. the insert also typically includes a tube extending upwards relative to the upper surface of the membrane and through the support structure, wherein when a negative pressure is applied to a distal end of the tube, a negative pressure is created in the sealed chamber of the cuvette, thereby creating a pressure gradient at the upper surface of the porous membrane.
According to another aspect of the invention, an electroporation cuvette insert is provided in combination with an electroporation cuvette. The insert typically includes a porous membrane having an upper surface and a lower surface, a support structure for holding the membrane, the support structure being of a sufficient dimension to securely fit within the inner walls of the electroporation cuvette, wherein the support structure creates a substantially sealed chamber within the cuvette below the lower surface of the membrane when inserted into the cuvette. the insert also typically includes a tube extending upwards relative to the upper surface of the membrane and through the support structure, wherein when a negative pressure is applied to a distal end of the tube, a negative pressure is created in the sealed chamber of the cuvette, thereby creating a pressure gradient at the upper surface of the porous membrane.
According to yet another aspect of the present invention, a method is provided for forming a fusion-based cuvette from an electroporation cuvette. The method typically includes providing an electroporation cuvette having external electrode contacts proximal a bottom portion, providing an insert having a support structure for holding a porous membrane, and a tube extending through the support structure, the insert being configured to fit within the electroporation cuvette, and securing the insert within the electroporation cuvette so as to form a substantially air-tight chamber below the support structure and membrane, and such that application of a vacuum to the end of the tube creates a negative pressure in the chamber and a pressure gradient at an upper surface of the membrane.
Reference to the remaining portions of the specification, including the drawings claims and Appendices, will realize other features and advantages of the present invention. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with respect to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements.
REFERENCES:
patent: 6150148 (2000-11-01), Nanda et al.
patent: 6221665 (2001-04-01), Jaroszeski et al.
Bio-Rad Laboratories, Inc.
Gray Gerald T.
Redding David A.
Townsend and Townsend / and Crew LLP
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