Method and apparatus for introducing biological substances into living cells

• US 5,204,253 A
• Filed: 05/29/1990
• Issued: 04/20/1993
• Est. Priority Date: 05/29/1990
• Status: Expired due to Term

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University), E. I. du Pont de Nemours and Company (Corteva, Inc.), Duke University
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Bruner, Ronald F., DeVit, Michael J., Johnston, Stephen A., Sanford, John C.
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/529,989
• Time in Patent Office: 1,057 Days
• Field of Search: 435/172.1-172.3, 435/240.2, 435/240.1, 435/284, 435/287, 935/52, 935/53, 935/85, 604/68-70, 124/61, 124/71, 124/73
• US Class Current: 435/459
• CPC Class Codes: C12M 35/00 Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87
• CPC Class Codes: C12M 35/04 Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
• CPC Class Codes: C12N 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

A process is described which uses a "cold" gas shock to accelerate microprojectiles wherein particles are presented to the gas shock on a planar surface perpendicular to the plane of expansion of the gas shock wave. Several different apparatus capable of accomplishing this method are described.

Claims

1. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet, and

exposing said sheet to the force of an instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said cold gas shock wave causing distension but not rupture of the membrane, thereby protecting the target from the shock wave.

2. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning a restraining screen between the target of cells and the membrane,

positioning particles carrying biological materials on the target side of the planar carrier sheet, and

exposing said sheet to the force of an instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said restraining screen being positioned such that the membrane is restrained against the screen prior to full distension or rupture in response to said cold gas shock wave, allowing the particles to pass through the screen to the target.

3. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet, and

exposing said sheet to the force of an instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said cold gas shock wave causing distension and rupture of the membrane, thereby launching, deagglomerating and dispersing the particles over a broad region of the target cells.

4. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a planar carrier sheet having a target side, said sheet being a non-resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet, and

exposing said sheet to the force of an instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said cold gas shock wave causing distension and rupture of the membrane, thereby launching, deagglomerating and dispersing the particles over a broad region of the target cells.

5. The method of claim 4 wherein the sheet is aluminum foil.

6. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a planar carrier sheet, said sheet being a screen,

providing a target of cells,

positioning particles carrying biological materials on the screen, and

exposing said screen to the force of an instantaneous cold gas shock wave so as to accelerate the particles to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said screen being positioned so as to permit the force of the shock wave to pass through it to accelerate the particles by gas entrainment.

7. The method of claim 6 which includes the additional step of baffling the shock wave after it passes the screen, to deflect some of the force of the shock wave away from the target cells.

8. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a source of pressurized gas and a rupturable membrane seal in communication with the source of gas,

providing a planar carrier sheet having a target side,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet,

rupturing the seal to catastrophically release the gas so as to generate an instantaneous cold gas shock wave,

exposing said sheet to the force of the instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells.

9. The method of claim 8 wherein the sheet is a resilient membrane fixedly secured at its edges against movement, and the cold gas shock wave causes distension but not rupture of the membrane, thereby protecting the target from the shock wave.

10. The method of claim 8 wherein the sheet is a resilient membrane fixedly secured at its edges against movement, and includes the step of positioning a restraining screen between the target cells and the membrane, such that the membrane is restrained against the screen prior to full distension or rupture, allowing the particles to be launched, passing through the screen to the target.

11. The method of claim 8 wherein the sheet is a resilient membrane fixedly secured at its edges against movement, and permitting the shock wave to cause distension and rupture of the membrane, thereby launching, deagglomerating and dispersing the particles over a broad region of the target cells.

12. The method of claim 8 further including positioning a barrier between the sheet and the target of cells, the sheet being positioned so as to be not tightly anchored in place, such that the sheet will be accelerated to move freely in the direction of the target cells until restrained by the barrier which permits the particles to move toward the target cells.

13. The method of claim 12 wherein the sheet is formed of polyimide or polyester film.

14. The method of claim 12 wherein the barrier is a screen in the path of movement of the sheet.

15. The method of claim 8 wherein the planar carrier sheet is a screen that permits the force of the shock wave to pass through it to accelerate the particles by gas entrainment.

16. The method of claim 15 which includes the additional step of baffling the shock wave after it passes the screen, to deflect some of the force of the shock wave away from the target cells.

17. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a source of pressurized gas,

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet,

rapidly releasing the source of gas so as to generate an instantaneous cold gas shock wave,

exposing said sheet to the force of the instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said cold gas shock wave causing distension but not rupture of the membrane, thereby protecting the target from the shock wave.

18. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a source of pressurized gas,

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning a restraining screen between the target of cells and the membrane,

positioning particles carrying biological materials on the target side of the planar carrier sheet,

rapidly releasing the source of gas so as to generate an instantaneous cold gas shock wave,

exposing said sheet to the force of the instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said restraining screen being positioned such that the membrane is restrained against the screen prior to full distension or rupture in response to said cold gas shock wave, allowing the particles to pass through the screen to the target.

19. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a source of pressurized gas,

providing a planar carrier sheet having a target side, said sheet being a resilient membrane fixedly secured at its edges against movement,

providing a target of cells,

positioning particles carrying biological materials on the target side of the planar carrier sheet,

rapidly releasing the source of gas so as to generate an instantaneous cold gas shock wave,

exposing said sheet to the force of the instantaneous cold gas shock wave so as to accelerate the particles free of the carrier sheet and to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said cold gas shock wave causing distension and rupture of the membrane, thereby launching, deagglomerating and dispersing the particles over a broad region of the target cells.

20. A method of introducing particles carrying biological materials into a target of cells, said method comprising the steps of:

providing a source of pressurized gas,

providing a planar carrier sheet, said sheet being a screen,

providing a target of cells,

positioning particles carrying biological materials on the screen, and

rapidly releasing the source of gas so as to generate an instantaneous cold gas shock wave,

exposing said sheet to the force of the instantaneous cold gas shock wave so as to accelerate the particles to cause them to penetrate the surface of the cells and become incorporated into the interior of the cells without killing the cells, said screen being positioned so as to permit the force of the shock wave to pass through it to accelerate the particles by gas entrainment.

21. The method of claim 20 which includes the additional step of baffling the shock wave after it passes the screen, to deflect some of the force of the shock wave away from the target cells.

22. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the portion of the pressure chamber toward the throat portion being sealed by a rupturable membrane.

23. The apparatus of claim 22 wherein the membrane is constructed of polyimide film.

24. The apparatus of claim 22 wherein the chamber includes means to mechanically rupture the membrane.

25. The apparatus of claim 24 wherein the high pressure chamber has a restricted inlet to limit the rate of flow of gas into the chamber.

26. The apparatus of claim 24 wherein the means to mechanically rupture the membrane is electromagnetically actuated.

27. The apparatus of claim 24 wherein the positioning means includes a disposable carrier sheet having a target side positioned in the throat portion and constructed so that the particles can be positioned on the target side of the carrier sheet, whereby the particles can be propelled by the force of the shock wave to the target.

28. The apparatus of claim 27 wherein the carrier sheet is resilient and which further includes anchoring means to secure the carrier sheet in the throat portion by gripping the periphery of the carrier sheet, the anchoring means being positionable at various locations along the axis.

29. The apparatus of claim 28 wherein the positioning means further includes a screen secured by its edges between the carrier sheet and the target, thereby to restrain the movement of the carrier sheet, but allowing the carrier sheet to distend partially in response to the shock wave.

30. The apparatus of claim 27 wherein the carrier sheet is rupturable in response to the shock wave and which includes anchoring means to secure the carrier sheet in the throat portion by gripping the periphery of the carrier sheet, the anchoring means being positionable at various locations along the axis.

31. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the positioning means includes a disposable carrier sheet having a target side positioned in the throat portion and constructed so that the particles can be positioned on the target side of the carrier sheet, whereby the particles can be propelled by the force of the shock wave to the target, the carrier sheet being resilient and which includes anchoring means to secure the carrier sheet in the throat portion by gripping the periphery of the carrier sheet, the anchoring means being positionable at various locations along the axis.

32. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the positioning means includes a disposable carrier sheet having a target side positioned in the throat portion and constructed so that the particles can be positioned on the target side of the carrier sheet, whereby the particles can be propelled by the force of the shock wave to the target, the carrier sheet being resilient and which includes anchoring means to secure the carrier sheet in the throat portion by gripping the periphery of the carrier sheet, the anchoring means being positionable at various locations along the axis, the positioning means further includes a screen secured by its edges between the carrier sheet and the target, thereby to restrain the movement of the carrier sheet, but allowing the carrier sheet to distend partially in response to the shock wave.

33. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the positioning means includes a disposable carrier sheet having a target side positioned in the throat portion and constructed so that the particles can be positioned on the target side of the carrier sheet, whereby the particles can be propelled by the force of the shock wave to the target, the carrier sheet being rupturable in response to the shock wave and which includes anchoring means to secure the carrier sheet in the throat portion by gripping the periphery of the carrier sheet, the anchoring means being positionable at various locations along the axis.

34. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the portion of the pressure chamber toward the throat portion being sealed by a rupturable membrane, the positioning means includes a second membrane secured to the throat portion by means for gripping the periphery of the second membrane, the second membrane being rupturable by the shock wave, a resilient carrier sheet having a target side positioned between the second membrane and the target, the carrier sheet is not securely anchored to the throat portion, such that it can fly freely, the carrier sheet further constructed so that particles can be positioned on the target side thereof, and an anchored barrier positioned between the resilient carrier sheet and the target and constructed so as to stop the carrier sheet but allow particles to continue to the target.

35. Apparatus for introducing particles carrying biological materials into a target of cells and/or tissue comprising:

a closed housing capable of sustaining a vacuum, the housing having a first port for applying a vacuum thereto, a major axis, and second and third ports lying on the axis, the third port defining a throat portion where particles are accelerated and the third port located on the target side of the housing,

a high pressure chamber positioned in the second port and having a portion positioned toward the throat portion, the pressure chamber constructed so as to release gas stored therein to provide a gas shock wave directed toward the throat portion,

means for positioning the particles in the throat portion for their acceleration by the force of the shock wave provided by the high pressure chamber through the third port toward the target, and an interface secured to the throat portion for coupling the throat portion to the target, the positioning means includes a disposable carrier sheet having a target side positioned in the throat portion and constructed so that the particles can be positioned on the target side of the carrier sheet, whereby the particles can be propelled by the force of the shock wave to the target, the interface further including a nozzle attached to the throat portion and constructed so as to pass the particles to a limited region of the target, and a screen positioned in the interface to prevent the target from being drawn into the interface.