Targeted system for removing tumor cells from cell populations

• US 6,143,535 A
• Filed: 03/27/1998
• Issued: 11/07/2000
• Est. Priority Date: 03/27/1997
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Bernhard O. Palsson
• Inventors: Palsson, Bernhard O.
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US09/049,677
• Time in Patent Office: 956 Days
• Field of Search: 435/173.1, 435/173.4, 435/173.7, 424/577
• US Class Current: 435/173.1
• CPC Class Codes: C12N 5/0087 Purging against subsets of blood cells, e.g. purging alloreactive T cells
• CPC Class Codes: C12N 5/0093 Purging against cancer cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G02B 21/0088 Inverse microscopes
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence

Abstract

A method is presented to remove contaminating tumor cells from a cell population. The method includes labeling individual tumor cells in the population and then killing them with a high energy laser beam. The laser is focused so that it specifically kills the identified tumor cell, but not the remaining cells in the population.

Claims

1. A method for eliminating a first population of cells from a second population of cells, comprising:

a) labeling said first population of cells so that said first population of cells can be distinguished from said second population of cells;

b) placing said first population of cells and said second population of cells is in a stationary position on a surface;

c) locating one cell in said first population of cells by reference to said label;

d) applying a first pulse from a controlled energy source to said one cell;

e) determining if said one cell was killed by said first pulse; and

f) applying a second pulse from a controlled energy source if said one cell was not killed.

2. The method of claim 1, wherein the method of labeling said first population of cells comprises labeling tumor cells.

3. The method of claim 1, wherein the method of eliminating a first population of cells from a second population of cells comprises eliminating a first population of tumor cells from a second population of non-tumor cells.

4. The method of claim 1, wherein the method of applying a first pulse comprises applying a laser pulse.

5. The method of claim 1, wherein the method of applying a second pulse comprises applying a laser pulse.

6. A method of eliminating tumor cells from within a cell population that includes non-tumor cells, comprising the steps of:

a) labeling said non-tumor cells so that the non-tumor cells can be distinguished from said tumor cells;

b) locating one of said tumor cells by identifying a non-labeled cell in said cell population; and

c) killing said tumor cell by applying a pulse from a controlled energy source to said located tumor cell while said tumor cell is in a substantially stationary position on a surface.

7. The method of claim 6, wherein the step of killing said tumor cell comprises applying a laser pulse.

8. The method of claim 6, wherein the step of killing said tumor cell comprises applying a controlled energy source to said located tumor cell while said tumor cell is in a stationary position on a nominally flat surface.

9. The method of claim 6, wherein the step of killing said tumor cell comprises applying a controlled energy source to a located tumor cell while said cell population is at confluence.

10. A method of killing a first population of cells within a mixture of cells, said mixture comprising said first population of cells and a second population of cells, comprising:

a) illuminating a portion of said mixture of cells, wherein said first population of cells or said second population of cells is labeled such that they are substantially distinguishable from one another;

b) capturing an image of a plurality of cells in the illuminated portion of said mixture of cells;

c) determining at least two dimensional coordinates of a cell of said first population of cells by reference to the captured image, and by reference to the presence of, or lack of, the label on said cell; and

d) applying a lethal dose of energy to the coordinates of said cell.

11. The method of claim 10, wherein said label is specific for said first population of cells.

12. The method of claim 10, wherein said label is specific for said second population of cells.

13. The method of claim 10, wherein said image is captured with a camera.

14. The method of claim 10, wherein said first population of cells is comprised of tumor cells, and said second population of cells is comprised of non-tumor cells.

15. The method of claim 10, wherein said lethal dose of energy is from a laser source.

16. The method of claim 15, wherein the wavelength of energy emitted from said laser source is approximately between 100 nanometers to 30 micrometers.

17. The method of claim 10, additionally comprising:

a) determining whether said cell was killed by said lethal dose of energy; and

b) applying a second lethal dose of energy to said cell if said cell was not killed.

18. The method of claim 10, wherein said mixture of cells is in a substantially stationary position.

19. The method of claim 10, wherein said mixture of cells is on a substantially flat surface.

20. The method of claim 10, wherein said mixture of cells is substantially confluent on a surface.

21. The method of claim 10, wherein said mixture of cells is present as a substantially single layer of cells on a surface.

22. The method of claim 10, wherein (c) and (d) are repeated for every cell in said first population of cells that is captured in said image.

23. The method of claim 10, wherein said first population of cells is comprised of tumor cells, and said second population of cells is comprised of hematopoietic cells.

24. The method of claim 23, wherein said hematopoietic cells are comprised of cells that have been enriched for hematopoietic stem cells.

25. The method of claim 24, wherein said hematopoietic stem cells are enriched based on expression of the CD34 surface protein.

26. The method of claim 24, wherein said hematopoietic stem cells are enriched based on the expression, or lack of expression, of one or more cell markers.

27. The method of claim 26, wherein said hematopoietic stem cells are enriched based on expression, or lack of expression, of a cell marker selected from the group consisting of:

Thy-1, CD38, CD15, CD11b, Gly-A, CD3, and CD19.

28. The method of claim 24, wherein said hematopoietic stem cells are enriched by using deep bed filtration or counterflow elutriation.

29. The method of claim 10, wherein said method of killing is at least partially performed under the control of a computer.

30. The method of claim 10, wherein said first population of cells is comprised of fibroblast cells, and said second population of cells is comprised of cartilage cells.

31. The method of claim 10, wherein said first population of cells is comprised of T cells.

32. The method of claim 10, wherein said first population of cells is comprised of non-tumor cells, and said second population of cells is comprised of tumor cells.