Devices and methods for isolating and recovering target cells

• US 20030175818A1
• Filed: 09/18/2002
• Published: 09/18/2003
• Est. Priority Date: 03/15/2002
• Status: Abandoned Application

• Current Assignee: Amelia A. Ross, Steve Bernstein
• Original Assignee: Amelia A. Ross, Steve Bernstein
• Inventors: Bernstein, Steve, Ross, Amelia A.
• Application Number:
  US10/247,223
• Publication Number:
  US 20030175818A1
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: C12M 23/46 Means for fastening
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

A cell isolating device and method is provided to concentrate or isolate cells with specific characteristics from a mixture of different cell types. One embodiment may comprise two subtypes of antibodies that are directly conjugated to biotin (Ab_b) and conjugated to a fluorescent molecule (Ab_f). The conjugated antibodies (Ab_b+Ab_f) bind to the target cells in a mixed cell suspension. The cell suspension is then passed over an immobilized avidin or streptavidin substrate on a glass microscope slide. The biotinylated target cells adhere to the avidin/streptavidin substrate, while the unbound cells are washed off and collected in a wicking member. Captured cells on the avidin/streptavidin substrate may then be visualized directly using a fluorescent microscope or detected and enumerated via an on-board fluorescent detection device. Additional chemicals and/or physical manipulation may then be applied to the device to release viable target cells for subsequent analysis.

Claims

1. A device for recovering a target cell from a cellular matrix, comprising:

a housing having at least one wall and a base, said base having a plurality of openings;

a reaction surface positioned proximate to said plurality of openings; and

a fluid absorbing media in communication with said substrate and adjacent to said housing.

2. The device of claim 1 wherein said housing further includes at least one fastening member capable of engaging said reaction surface.

3. The device of claim 1 wherein said housing and said reaction surface are in spaced relationship.

4. The device of claim 3 wherein said reaction surface and said housing are separated by a distance of approximately 2 to 20 microns.

5. The device of claim 1 wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.

6. The device of claim 5 wherein said bioactive coating has a surface area of approximately 8 cm².

29. A method of recovering viable target cells from a cellular mixture, comprising:

providing an apparatus of claim 1;

exposing the cellular mixture to a targeting solution to form a target cell mixture;

adding the target cell mixture to the apparatus;

controlling the rate of contact between the reaction surface and the target cell mixture by establishing a predetermined delivery distance for said target cell mixture to traverse to the reaction surface;

analyzing the reaction surface for target cells; and

removing target cells from the reaction surface by delivering a releasing solution to the reaction surface; and

collecting the releasing solution, wherein the releasing solution includes the target cells.

7. A method of recovering a target cell from a cellular mixture comprising:

exposing a cellular mixture to a targeting solution to form a target cell mixture;

delivering said target cell mixture to a reaction surface;

controlling the delivery of said target cell mixture so as to optimize the flow over said reaction surface;

retaining said target cells on said reaction surface;

delivering a releasing solution to said reaction surface; and

collecting said releasing solution, wherein said releasing solution includes said target cells.

8. A method as set forth in claim 7, wherein the controlling of the delivery of the target cell mixture is performed by establishing a predetermined delivery distance for said target cell solution to traverse to said reaction surface.

9. A method as set forth in claim 8 wherein the predetermined delivery distance is approximately 2 to 20 microns.

10. A method as set forth in claim 8, wherein the controlling the delivery of the target cell mixture further includes absorbing non-reacting amounts of said target cell solution from said reaction surface at a predetermined rate.

11. The method of claim 10 further comprising delivering another aliquot of the target cell releasing solution to said reaction surface, mechanically agitating said target cell releasing solution, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.

12. The method of claim 7 wherein said targeting solution includes a marker compound comprising a binder molecule and a probe.

13. The method of claim 12 wherein said binder molecule is selected from the group consisting of antibodies, antigens, glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids, sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes.

14. The method of claim 13 wherein said probe is selected from the group consisting of flurochromes, radiolabels, fluorescent agents, and chromophores.

15. The method of claim 7 wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.

16. The method of claim 7 wherein said releasing solution is a buffer solution having a pH ranging from about 3.0 to 6.0.

17. The method of claim 7 wherein said releasing solution includes a cell releasing agent selected from the group consisting of chymopapain, trypsin, chymotrypsin, V8 protease, Ethylenediaminetetraacetic Acid, cyanogen bromide, or 2-Nitro-5-thiocyanobenzoate.

18. A method of isolating target cells from a cellular mixture comprising:

exposing a cellular mixture to a targeting solution to form a target cell mixture;

delivering said target cell mixture to a reaction surface;

controlling the delivery of said target cell mixture so as to optimize the flow over said reaction surface;

retaining said target cells on said reaction surface;

analyzing said target cells remaining on said reaction surface; and

delivering at least one target cell releasing solution to said reaction surface, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.

19. A method as set forth in claim 18, wherein the controlling of the delivery of the target cell mixture is performed by establishing a predetermined delivery distance for said target cell solution to traverse to said reaction surface.

20. A method as set forth in claim 19 wherein the predetermined delivery distance is approximately 2 to 20 microns.

21. A method as set forth in claim 19, wherein the controlling the delivery of the target cell mixture further includes absorbing non-reacting amounts of said target cell solution from said reaction surface at a predetermined rate.

22. A method as set forth in claim 21 further comprising delivering another aliquot of the target cell releasing solution to said reaction surface, mechanically agitating said target cell releasing solution, and collecting said target cell releasing solution, wherein said target cell releasing solution includes said target cells.

23. The method of claim 18 wherein said targeting solution includes a marker compound comprising a binder molecule and a probe.

24. The method of claim 23 wherein said binder molecule is selected from the group consisting of antibodies, antigens, glycoproteins, lectins, hormones, cell receptors, vitamins, amino acids, sugars, lipids, fatty acids, liposomes, DNA probes, and RNA probes.

25. The method of claim 24 wherein said probe is selected from the group consisting of flurochromes, radiolabels, fluorescent agents, and chromophores.

26. The method of claim 18 wherein said reaction surface comprises a bioactive coating selected from the group consisting of avidin, streptavidin, avidin derivatives, streptavidin derivatives, and blends thereof.

27. The method of claim 18 wherein said target cell releasing solution is a buffer solution having a pH ranging from about 3.0 to 6.0.

28. The method of claim 18 wherein said releasing solution includes a cell releasing agent selected from the group consisting of chymopapain, trypsin, chymotrypsin, V8 protease, Ethylenediaminetetraacetic Acid, cyanogen bromide, or 2-Nitro-5-thiocyanobenzoate.