Process and apparatus for cell sorting

• US 5,914,262 A
• Filed: 06/21/1996
• Issued: 06/22/1999
• Est. Priority Date: 05/21/1993
• Status: Expired due to Term

• Current Assignee: Smithkline Beecham PLC
• Original Assignee: Smithkline Beecham PLC
• Inventors: Drake, David, Stovold, John Spencer, Divers, Moira Sloan, Addicott, John Kenneth, Arlington, Stephen Adrian, MacMichael, Donald Bruce Atherton, Normansell, Ian David
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Swartz, Rodney P.
• Application Number:
  US08/556,920
• Time in Patent Office: 1,096 Days
• Field of Search: 209/3.1, 209/511, 209/552, 209/576, 356/73, 435/177, 435/178, 435/243, 424/93, 424/493
• US Class Current: 435/243
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2035/00574 Means for distributing beads

Abstract

A method of distinguishing between microorganisms having different attributes comprises the steps of: encapsulating one or more cells within a polymeric bead; incubating the cell(s) within the bead in a growth-supporting medium for a time period sufficient for multiplication of viable cells within the bead; then non-invasively physically examining the bead or the adjacent vicinity of the bead for physical characteristics associated with the differing attributes of cells within the bead. Various forms of apparatus for carrying out the process are descried.

Claims

1. An apparatus for separating physically distinguishable polymeric beads at least some of which contain encapsulated cells possessing a selected attribute, comprising:

a vessel containing a suspension of the polymeric beads, at least some of which contain encapsulated cells possessing a selected attribute, in a suspension fluid;

means for presenting the beads to a detection means which is adapted to non-invasively physically detect and distinguish individual beads into classes based upon physical characteristics of the beads or the suspension fluid in the adjacent vicinity of the beads;

particle directing means adapted to physically separate beads of one of said classes from those of another class, and;

control means adapted to control the bead directing means in response to the output of the said detection means so as to direct each bead to a defined destination.

2. An apparatus according to claim 1 wherein the beads are transported and/or presented to the detection means in a flow of the suspension fluid.

3. An apparatus according to claim 2 wherein the means for presenting the beads to a detection means comprises means for channeling the suspension into one or more flow streams, in which streams the beads are individually serially disposed, the flow streams being directed into the vicinity of the detection means.

4. An apparatus according to claim 3 wherein the means for channeling the suspension into a flow stream in which the beads are individually serially disposed and comprise one or more flow tubes having a bore less than 2 bead diameters.

5. An apparatus according to claim 4 wherein downstream of the detecting and distinguishing means the flow tube terminates at an open end, this open end being opposite a facing entrance opening in a bead catcher, so that a bead may travel from the open end of the flow tube into the entrance opening of the bead catcher across the gap between the open end into the entrance opening, the open end of the flow tube and/or the entrance opening being provided with flow control means to allow or prevent passage of a bead from the open end of the flow tube to the said entrance opening.

6. An apparatus according to claim 5 wherein the entrance opening of the bead catcher, and/or the open end of the flow tube are moveable relative to each other in and out of a facing relationship so that beads may pass from the flow tube into the bead catcher when they are in the opposite facing relationship, but may not when they are out of the opposite facing relationship.

7. An apparatus according to claim 6 wherein the open end of the flow tube and the entrance opening in the bead catcher are both within an area of positive atmospheric pressure, and the bead catcher communicates with an area of lower atmospheric pressure.

8. An apparatus according to claim 4 wherein the flow tube is divided into a "Y" or otherwise forked tube arrangement with the flow of suspension along the stem towards the fork, with the detection means, monitoring a part of this stem, and provided with diverter means to divert selected beads traveling along the stem toward the fork along selected limbs of the fork one or more of the said limbs thereby comprising a bead catcher.

9. An apparatus according to claim 5 wherein the bead catcher communicates with a bead director to direct caught beads to a collector.

10. An apparatus according to claim 9 wherein the bead director comprises a downstream extension to the bead catcher along which particles may be directed by a positive atmospheric pressure difference along the extension.

11. An apparatus according to claim 10 wherein a region of the extension downstream of the open end of the bead catcher and upstream of the end of the extension is surrounded by a partial vacuum manifold.

12. An apparatus according to claim 1 wherein the detection means is an optical detection means.

13. A method for distinguishing between cells having different attributes, comprising;

encapsulating one or more cells from the culture within a polymeric bead;

incubating the cells within the bead in a growth-supporting medium for a time period sufficient for multiplication of viable cells within the bead;

then separating beads containing the biomass of multiplied viable cells having a selected attribute from beads which are empty or contain cells which do not have the selected attribute, by using the apparatus of claim 1.

14. An apparatus for separating physically distinguishable polymeric beads, comprising:

means for suspending the polymeric beads in a suspension liquid and to form a suspension of the beads in the suspension fluid,

means for channeling the suspension of beads into one or more flow tubes each having a bore less than two bead diameters and in which streams the beads are individually serially disposed, the flow streams being directed into the vicinity of the detection means,

means for presenting the beads to a detection means which is adapted to non-invasively physically detect and distinguish individual beads into classes based upon physical characteristics of the beads or the adjacent vicinity of the beads;

bead directing means adapted to physically separate beads of one of said classes from those of another class, and;

control means adapted to control the bead directing means in response to the output of the said detection means so as to direct each bead to a defined destination.

15. An apparatus for separating physically distinguishable polymeric beads, comprising:

means for suspending the polymeric beads in a suspension liquid and to form a suspension of the beads in the suspension fluid,

a detection means which is adapted to non-invasively physically detect and distinguish individual beads into classes based upon physical characteristics of the beads or the suspension fluid in the adjacent vicinity of the beads,

means for channeling the suspension of beads into one or more flow tubes in which streams the beads are individually serially disposed, the streams being directed to the detection means,

means for presenting the beads to the detection means;

downstream of the detecting and distinguishing means the flow tube terminating at an open end;

a bead catcher having an entrance opening in a facing relationship to said open end, so that a bead may travel from the open end of the flow tube into the entrance opening of the bead catcher,

flow control means provided at the open end of the flow tube and/or the entrance opening of the bead catcher to allow or prevent passage of a bead from the open end of the flow tube to the said entrance opening,

a bead director tube in communication with the entrance opening of the particle catcher, the entrance opening of the bead catcher being at a higher atmospheric pressure relative to the bead director tube such that beads entering the entrance opening of the bead catcher are directed by the difference in relative air pressure along the bead director tube.