Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2001-02-08
2003-11-11
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S285200, C435S286100, C435S286500, C435S288500, C204S164000, C204S403010, C204S409000
Reexamination Certificate
active
06645757
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to cell manipulation and transformation, and more particularly to a microelectromechanical (MEM) apparatus and method for transforming one or more living host cells by temporarily disrupting the integrity of the host cell so that particular substances of interest (e.g. molecules or macromolecules including DNA) can be introduced therein.
BACKGROUND OF THE INVENTION
Biologists often need to introduce into living host cells a wide range of substances which are normally excluded from the cell by the cell walls and outer cell membranes. One important application of cell manipulation is the introduction of genetic material into host cells for the purpose of genetic engineering. In this way, genetically-altered organisms can be produced for use in research, for synthesizing particular bioengineered proteins (e.g. insulin) or for producing crops having particular genetic features (e.g. resistance to pests). For these and other applications, particular biological stains, proteins, nucleic acids, organelles, chromosomes, nuclei, etc. can be introduced into biological cells.
Existing technologies for transporting genetic material into living cells involve uptake mechanisms, cell fusion, electroporation and microinjection. Uptake mechanisms generally involve suspensions of single cells, from which any existing cell wall materials have been removed enzymatically. Cell fusion incorporates genetic material into a cell by allowing that cell to fuse with another cell containing the genetic material of interest. Electroporation utilizes high electric fields to create pores in cells without causing permanent damage to the cells. Microinjection employs an extremely fine, drawn-out capillary tube (also termed a micropipette) which is used as a syringe needle to directly inject a biological substance an individual cell.
The present invention represents an improvement over the prior art by providing an apparatus and method that allows individual host cells to be transformed one at a time under carefully controlled circumstances, and in a controlled environment.
An advantage of the present invention is that host cells can be moved one at a time through a flow channel wherein each host cell can be transformed by temporarily disrupting the cell wall or membrane in a controlled and reproducible way to allow a particular substance of interest to enter into the cell, thereby transforming the cell.
Another advantage of the present invention is that the cell transformation can be performed without the need for any tedious and difficult manual alignment or manipulation.
A further advantage of the present invention is that the cell transformation can be accomplished using electrical or mechanical poration, or a combination of both.
These and other advantages of the present invention will become evident to those skilled in the art.
SUMMARY OF THE INVENTION
The present invention relates to an apparatus for transforming a host cell, comprising a flow channel lined with silicon nitride for containing the host cell and a surrounding fluid; and means, located at least partially within the flow channel, for temporarily disrupting the integrity of the host cell and thereby introducing a substance into the host cell for transforming the host cell. The flow channel and the cell disrupting means can be formed on a common substrate which generally comprises silicon.
The cell disrupting means can comprise either a pair of electrodes located proximate to the host cell on opposite sides of the flow channel for generating an electric field across the host cell in response to a voltage applied between the pair of electrodes, or a moveable member located on one side of the flow channel for mechanically abrading, impacting or penetrating the host cell. The electrodes and the moveable member can be pointed (e.g. with a single solid or hollow point, or a plurality of points or sharp edges arranged as corrugations or serrations facing the host cell). The moveable member is operatively connected to an electrostatic actuator formed on the substrate (e.g. through a compliant structure) to provide for movement thereof. This movement can be a single motion (e.g. for penetrating the host cell, or for moving into position to constrict the channel and abrade the host cell as the cell is urged along the constricted channel) or reciprocating motion (e.g. for repeatedly pricking or irritating the cell wall or membrane).
The flow channel preferably includes means for urging the host cell to move along the flow channel for proper location or alignment of the host cell prior to or during disruption of the cell membrane. This can be done, for example, by generating a pressure gradient in the fluid within the flow channel using an external pump, or by providing a plurality of electrodes spaced along the length of the flow channel to generate an electroosmotic or electrokinetic force for moving the fluid based on a voltage applied between the spaced electrodes.
Input and output ports can be connected to the ends of the flow channel for transferring the host cell and the fluid into and out from the channel. An optional third port can also be provided that is in fluid communication with the flow channel so that the cell-transforming substance (i.e. a biological stain, a protein, nucleic acid, antibody, organelle, chromosome, nuclei, virus, plasmid, bacteria, etc., which is of interest for use in transforming the host cell) can be introduced into the fluid, or directly into the host cell.
The present invention also relates to an apparatus for transforming a host cell that comprises a flow channel formed on a substrate for isolating the host cell from a plurality of other host cells within a fluid surrounding the host cell; and a moveable member formed on the substrate and located on one side of the flow channel for abrading, impacting or penetrating the host cell for transferring a substance into the host cell and thereby transforming the host cell. The moveable member is operatively connected to an electrostatic actuator located on the substrate for providing motion to the moveable member. This connection can be through a linkage which comprises a compliant structure that provides a stroke for the moveable member that is generally larger than the stroke provided by the electrostatic actuator. An end of the moveable member that abrades, impacts or penetrates the host cell can be pointed or serrated. An optional stationary member can be located opposite the moveable member, with the stationary member generally being pointed or serrated on a side thereof facing the host cell. In some embodiments of the present invention, the moveable and stationary members can form electrodes for applying a voltage across the host cell to further condition (e.g. by electroporation) the host cell for receiving the substance to be transferred therein.
The flow channel generally comprises polysilicon (i.e. polycrystalline silicon) and can be lined with silicon nitride for biocompatibility. The flow channel can further include a plurality of electrodes spaced along the length of the flow channel for generating a flow of the fluid therein in response to a voltage applied between the electrodes. This fluid flow can be used to urge the host cell along the flow channel and to align the host cell with the moveable member. The flow channel also generally includes input and output ports as described previously, and can further include an optional third port for admitting the substance to be transferred to the host cell into the fluid, or directly into the host cell (e.g. through a conduit formed in the moveable member).
The present invention further relates to an apparatus for transforming a host cell, that comprises a flow channel for isolating the host cell from a plurality of other host cells in a fluid, with the fluid further including a substance to be transferred into the host cell for transforming the host cell; a stationary electrode located on one side of the flow channel; and a moveable electrode located o
Galambos Paul C.
Okandan Murat
Hohimer John P.
Redding David A.
Sandia Corporation
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