Sterile flow cytometer and sorter with mechanical isolation between flow chamber and sterile enclosure and methods for using same

• US 5,776,781 A
• Filed: 02/20/1997
• Issued: 07/07/1998
• Est. Priority Date: 04/25/1995
• Status: Expired due to Term

• Current Assignee: Novartis Ag
• Original Assignee: Systemax Incorporated
• Inventors: Lewis, Hugh, Vardanega, Michael H., Tichenor, Edie, Medeiros, David J., Joubran, John, Swan, Raymond
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US08/803,439
• Time in Patent Office: 502 Days
• Field of Search: 422/33, 422/28, 422/82.01, 422/82.05, 422/104, 422/295, 436/52, 436/63, 436/164, 356/72, 356/73, 250/461.2, 209/3.1
• US Class Current: 436/63
• CPC Class Codes: B01L 1/04 Dust-free rooms or enclosures
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2035/00277 Special precautions to avoid contamination (e.g. enclosures, glove- boxes, sealed sample carriers, disposal of contaminated material)

Abstract

An apparatus and process for measuring particles that prevents the particles from being contaminated during the measuring and optional sorting process wherein a flow chamber or region is mechanically isolated from a sterile enclosure which surrounds the flow chamber. The present invention in one embodiment provides an apparatus comprising of a droplet generator, a charging system, a deflecting system, a sterilization system, a light source, a detector, a cooling system and a vacuum system. The droplet generator and the charging system and the flow chamber associated with the droplet generator operate within an illumination frame where the particles (e.g. biological cells) are actually measured. In order to prevent the biological cells from being contaminated, the illumination frame is located within an isolation chamber which provides an environment that can be sterilized before and after the actual measuring and sorting of the cells, but the illumination frame is mechanically isolated from the isolation chamber. The sterilization system, located outside the isolation chamber sterilizes the isolation chamber before cells are measured and sorted within the isolation chamber, and furthermore, ensures that the isolation chamber is maintained at a pressure above (or alternatively, below) atmospheric pressure during the measuring and sorting of the biological cells. The illumination frame, the light source and the detector are supported by a table to enable the light beam(s) from the light source to intersect the biological cells flowing through the illumination frame, thereby emitting flashes of light (or otherwise effecting the light beam) which are detected by the detector.

Claims

1. A sterile system for measuring at least one property of a sample of cells, said sterile system comprising:

an isolation chamber for providing an enclosed sterile environment;

a sterilization system coupled to said isolation chamber, said sterilization system sterilizing an area located within said isolation chamber;

a pressurization system coupled to said isolation chamber, said pressurization system pressurizing said isolation chamber during said measuring of at least one property such that said pressure in said isolation chamber is not substantially equivalent to atmospheric pressure;

a cell station for holding said sample of cells;

an illumination frame located within said isolation chamber, said illumination frame being coupled to said cell station wherein said cell station provides said illumination frame with a stream of cells from said sample of cells;

a light source coupled to said illumination frame wherein said light source provides said illumination frame with at least one beam of light for measurement of said at least one property;

a detector coupled to said illumination frame, said detector collects light from said stream of cells flowing through said illumination frame, said detector further transforms said light into electrical pulses to be measured.

2. The sterile system as in claim 1, wherein said at least one property of said sample of cells comprises a cellular constituent and wherein said cell station is pressurized with a gas to drive said sample of cells from said cell station to said illumination frame.

3. The sterile system as in claim 1, wherein said at least one property of said sample of cells to be measured is labeled with a specific type of fluorescent molecule wherein said specific type of fluorescent molecule binds to each cell within said sample of cells having said at least one property to be measured, said specific type of fluorescent molecule emits a light having an intensity proportional to said specific type of fluorescent molecule when said at least one beam of light from said light source intersects each of said cells within said sample of cells having said fluorescent molecule bound to it and wherein said pressure in said isolation chamber exceeds atmospheric pressure.

4. The sterile system as in claim 1, wherein said sterilization system injects a sterilant into said isolation chamber to sterilize an area located within said isolation chamber by increasing a concentration of said sterilant within said isolation chamber to an effective level, said sterilization system further circulates filtered air in said isolation chamber to ventilate said area located within said isolation chamber thereby reducing said concentration of said sterilant to a negligible level.

5. The sterile system as in claim 1, wherein said sterilization system comprises

a vapor hydrogen peroxide generator having an output coupled to an input of said isolation chamber, said vapor hydrogen peroxide generator injects hydrogen peroxide vapor into said isolation chamber to sterilize said isolation chamber, said vapor hydrogen peroxide generator further having an input coupled to an output of said isolation chamber, said vapor hydrogen peroxide generator ventilates said isolation chamber by returning said hydrogen peroxide vapor which has been previously injected into said isolation chamber back to said vapor hydrogen peroxide generator, wherein said isolation chamber is sterilized and ventilated before measuring at least one property of said sample of cells.

6. The sterile system as in claim 1, wherein said cell station has a first input and a second input and an output, said first input coupled to receive said sample of cells and said output is coupled to a first input of a droplet generator, said second input receives filtered air wherein said filtered air pressurizes said sample of cells to flow out of said cell station.

7. The sterile system as in claim 1, wherein said pressurization system includes a pressure regulator having an input coupled to a pressure sensor within said isolation chamber wherein said pressure sensor measures air pressure within said isolation chamber, said pressure regulator further has an output coupled to an input of a fan wherein said pressure regulator regulates the amount of filtered air entering said isolation chamber by regulating a speed of said fan, said fan further having a second input and an output wherein said second input is coupled to the atmosphere and said output is coupled to said input of said isolation chamber, said pressure regulator increases said air pressure within said isolation chamber by increasing said speed of said fan such that said fan draws air from said second input into said isolation chamber and said pressure regulator decreases said air pressure within said isolation chamber by decreasing said speed of said fan, and wherein air within said isolation chamber exits said sterilization system through an exhaust port which is coupled to said output of said isolation chamber.

8. The sterile system as in claim 1, wherein said isolation chamber includes a door, said door may be opened to move items in and out of said isolation chamber and may be sealed shut to provide an enclosed environment inside said isolation chamber which can be sterilized, said door includes a plurality of glove ports wherein an operator located outside said sterile system may handle objects within said isolation chamber without contaminating said sterile system.

9. The sterile system as in claim 1, said isolation chamber includes a transfer port having a first part and a second part linked by a locking mechanism wherein said second part is a canister and wherein objects located outside said isolation chamber may be placed inside second part and then placed inside said isolation chamber without breaking sterility.

10. The sterile system as in claim 1, wherein said isolation chamber includes a portion of a wall of said isolation chamber and wherein a first laser safety pipe and a second laser safety pipe are optically coupled to said isolation chamber at said portion.

11. The sterile system as in claim 1, wherein said isolation chamber includes a first wall and a second wall and wherein said light source provides a first light beam and a second light beam, said first light beam and said second light beam having a first angle between said light beams, said first light beam being directed through said first wall and said second light beam being directed through said second wall, and wherein said first wall and said second wall having a second angle between said walls which substantially equals said first angle.

12. The sterile system as in claim 1, wherein said isolation chamber includes a mesh basket for storing items within said isolation chamber.

13. The sterile system as in claim 1, further comprising a table having a first surface wherein said isolation chamber is positioned on said first surface of said table,

said isolation chamber further having a second surface wherein said illumination frame is positioned above said second surface of said isolation chamber,

said illumination frame being coupled to said table through an opening in said isolation chamber, said opening having a flexible seal between said illumination frame and said isolation chamber.

14. The sterile system as in claim 1, further comprising a table having a first surface wherein said isolation chamber is positioned above said first surface of said table, said isolation chamber having a first section and a second section wherein said first section is coupled to said second section, said first section extends beyond said first surface of said table such that said first section overhangs said table and said second section is secured to said first surface of said table.

15. The sterile system as in claim 1 further comprising electrically controlled valves for controlling flow of fluids and gas pressure in tubes, said tubes being located in said isolation chamber.

16. The sterile system as in claim 1, further comprising a cooling system within said isolation chamber wherein said cooling system includes a cooling line, a first cooling block and a second cooling block,

said cooling line provides a channel for a cooling mixture to flow through a portion of said first cooling block and a portion of said second cooling block wherein said cooling mixture cools said first cooling block and said second cooling block to a temperature that will keep cells within said sample of cells alive,

said first cooling block cools said sample of cells within said cell station,

said second cooling block cools said sample of cells within a cell collector.

17. The sterile system as in claim 16, wherein said first cooling block in said cooling system includes a cavity wherein a portion of said cell station is located inside said cavity, said first cooling block includes a transparent portion wherein the contents of said portion of said cell station located inside said cavity is visible from outside said isolation chamber.

18. The sterile system as in claim 1, wherein said illumination frame comprises a droplet generator, a charging system, a deflecting system and a cell collector wherein an output of said droplet generator is coupled to said charging system and an output of said charging system is coupled to said deflecting system and an output of said deflecting system is coupled to an input of said cell collector.

19. The sterile system as in claim 18, wherein said droplet generator in said illumination frame includes a flow chamber having a first input and a second input, said first input is coupled to an output of said cell station wherein said first input receives said stream of cells from said sample of cells, said second input is coupled to an output of a pressurized container wherein said second input receives a sheath fluid, each cell within said stream of cells flowing through said flow chamber is suspended in said sheath fluid wherein each of said cells within said stream of cells flows in a substantially single file through said flow chamber and wherein said substantially single file intersects at least one said beam of light generated by said light source;

said droplet generator further includes a nozzle wherein said nozzle receives said substantially single file of said sample of cells suspended in said jet of sheath fluid and said output of said nozzle generates a procession of uniform droplets falling at a constant rate wherein almost each cell within said sample of cells is isolated into a separate droplet.

20. The sterile system as in claim 19, further comprising a vacuum system wherein said vacuum system is coupled to said droplet generator near said output of said nozzle, said vacuum system removes debris from said droplet generator into a waste tank located outside said isolation chamber thereby preventing debris from clogging said nozzle.

21. The sterile system as in claim 19, wherein said illumination frame comprises a charging system wherein said charging system is capable of selectively charging each of said separate droplets falling from said nozzle of said droplet generator by establishing a charge at the surface of an unbroken column of a jet of sheath fluid extending from said nozzle of said droplet generator before said unbroken column of said jet of sheath fluid separates into said procession of uniform droplets.

22. The sterile system as in claim 21, wherein said illumination frame comprises said deflecting system including two charged deflection plates that establish a constant transverse electrostatic deflection field in which said procession of droplets passes.

23. The sterile system as in claim 22, wherein said cell collector comprises a plurality of collection receptacles such that said deflecting system can deflect each droplet in said procession of droplets into a specific collection receptacle.

24. The sterile system as in claim 23, wherein said cell collector is further coupled to a vacuum system wherein said vacuum system removes debris from at least one collection receptacle of said plurality of collection receptacles into said waste tank located outside said isolation chamber.

25. A method of measuring cells in a sterile environment, said method comprising the steps of:

sterilizing an isolation chamber;

maintaining said isolation chamber at a pressure not substantially equivalent to atmospheric pressure while sorting cells;

providing a stream of sample cells to a droplet generator wherein said sample cells flow through said droplet generator;

generating at least one beam of light from a light source wherein said beam of light intersects said stream of sample cells;

measuring at least one property from said stream of sample cells.

26. The method of measuring cells in said sterile environment as in claim 25 wherein said step of sterilizing said isolation chamber comprises the steps of:

sealing said isolation chamber to prevent contaminants from entering said isolation chamber;

closing a first valve to prevent a pressure regulator from injecting filtered air into said isolation chamber;

opening a second valve to provide a path from a vapor hydrogen peroxide generator to said isolation chamber such that said vapor hydrogen peroxide generator can inject filtered air and/or hydrogen peroxide vapor into said isolation chamber;

dehumidifying said isolation chamber by injecting filtered air from said vapor hydrogen peroxide generator into said isolation chamber;

sterilizing said isolation chamber by injecting hydrogen peroxide vapor into said isolation chamber, wherein said hydrogen peroxide vapor is contained within said isolation chamber until an area within said isolation chamber is sterilized;

aerating said isolation chamber by injecting filtered air into said isolation chamber.

27. The method of measuring cells in said sterile environment as in claim 25 wherein said step of providing a stream of sample cells to a droplet generator comprises supplying pressurized gas to a cell station coupled to said droplet generator, said pressuized gas driving said sample of cells contained in said cell station to said droplet generator.

28. A method as in claim 25 further comprising:

isolating mechanically said isolation chamber from an illumination frame, said illumination frame supporting said droplet generator.

29. A method as in claim 25 further comprising:

separating said stream of sample cells into a procession of droplets;

charging at least one droplet generated by said droplet generator;

deflecting said at least one droplet passing a deflecting system into a specific collection receptacle.

30. A method as in claim 25 wherein said pressure is above atmospheric pressure.

31. A method as in claim 25 further comprising:

sterilizing said isolation chamber after measuring said at least one property.

32. A method as in claim 25 wherein said isolation chamber includes a first wall and a second wall and wherein a light source provides a first light beam and a second light beam, said first light beam and said second light beam having a first angle between said light beams, said first light beam being directed through said first wall and said second light beam being directed through said second wall, and wherein said first wall and said second wall having a second angle between said walls which substantially equals said first angle.

33. A method as in claim 32 wherein said light source comprises two lasers.

34. A method of sorting cells in a sterile environment, said method comprising:

sterilizing an isolation chamber;

maintaining said isolation chamber at a pressure not substantially equivalent to atmospheric pressure;

providing a stream of sample cells to a droplet generator wherein said stream of sample cells include at least one normal cell, said stream of sample cells flow through said droplet generator;

generating at least one beam of light from a light source wherein said beam of light intersects said stream of sample cells;

measuring at least one property of at least one cell from said stream of sample cells;

separating said stream of sample cells into a procession of droplets wherein almost each said sample cell is isolated into a separate droplet;

charging at least one droplet generated by said droplet generator;

deflecting said at least one droplet into a specific collection receptacle such that said at least one normal cell is separated from a plurality of non-normal cells within said stream of sample cells.

35. The method of sorting cells in said sterile environment as in claim 34 wherein said at least one normal cell is a hematopoietic stem cell and said plurality of non-normal cells are not hematopoietic stem cells.

36. A method as in claim 34 wherein said step of providing a stream of sample cells to a droplet generator supplying pressurized gas to a cell station coupled to said droplet generator, said pressurized gas driving said sample cells contained in said cell station in said droplet generator.

37. A method as in claim 34 further comprising:

isolating mechanically said isolation chamber from an illumination frame, said illumination frame supporting said droplet generator.

38. A method as in claim 34 wherein said isolation chamber includes a first wall and a second wall and wherein a light source provides a first light beam and a second light beam, said first light beam and said second light beam having a first angle between said light beams, said first light beam being directed through said first wall and said second light beam being directed through said second wall, and wherein said first wall and said second wall having a second angle between said walls which substantially equals said first angle.

39. A method as in claim 34 further comprising:

sterilizing said isolation chamber after separating said at least one normal cell from said plurality of non-normal cells.

40. A method as in claim 34 wherein said pressure is above atmospheric pressure.

41. A method as in claim 34 wherein said pressure is below atmospheric pressure.