Optical selection of cells

• US 10,077,463 B2
• Filed: 01/14/2016
• Issued: 09/18/2018
• Est. Priority Date: 01/15/2015
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Cohen, Adam E., Chien, Miao-Ping
• Primary Examiner(s): Yamasaki, Robert J
• Assistant Examiner(s): Constantine, Charles Z
• Application Number:
  US14/995,716
• Publication Number:
  US 20160208308A1
• Time in Patent Office: 978 Days
• Field of Search: None
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C09B 23/00 Methine or polymethine dyes, e.g. cyanine dyes
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C12N 5/0006 Modification of the membrane of cells, e.g. cell decoration
• CPC Class Codes: C12N 5/0081 Purging biological preparations of unwanted cells
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/5026 on cell morphology
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Provided herein are photochemical crosslinkers and photocleavable crosslinkers and their uses in methods for cell selection from cell cultures. The photochemical crosslinkers comprise a fluorescent dye and a radical generator. The photocleavable crosslinkers comprise a photocleavable linker linking two electrophilic groups to each other. Also provided are systems for imaging cells comprising a plurality of cells crosslinked to extracellular matrix proteins using a crosslinker as described, an imaging apparatus, an illuminating apparatus, and software for image processing.

Claims

1. A method of selecting cells with features of interest comprising:

a) providing a culture of cells in a culture dish;

b) imaging the cells to identify a cell of interest;

c) adding a photochemical crosslinker comprising a fluorescent dye linked to a radical generator to the culture of cells; and

d) illuminating the cell of interest with light so that the radical generator produces a radical which covalently cross-links the cell of interest to the surface of the culture dish.

2. The method of claim 1 further comprising washing away cells that are not attached to the surface of the culture dish through the crosslinker.

3. The method of claim 2, wherein the step of washing away cells comprises incubating the culture of cells with an enzyme.

4. The method of claim 3, wherein the enzyme is a proteolytic enzyme.

5. The method of claim 3, wherein the enzyme is an enzyme of marine origin having proteolytic and collagenolytic activity.

6. The method of claim 3, wherein the enzyme istrypsin.

7. The method of claim 1 further comprising isolating the cell of interest from the culture dish.

8. The method of claim 1, wherein the culture dish is coated with cell adhesion proteins.

9. The method of claim 8, wherein the cell adhesion protein is an extracellular matrix protein.

10. The method of claim 9, wherein the extracellular matrix protein is fibronectin, collagen, laminin, fibrillin, vitronectin, thrombospondins, tenascins, entactins (or nidogens), nephronectin, or fibrinogen, osteopontin, agrin, aggrecan, decorin, F-Spondin, matrix extracellular phosphoglycoprotein (MEPE), nidogen-1, testican, poly-L-lysine, poly-D-lysine, poly-L-orinthine, or a combination thereof.

11. The method of claim 1, wherein the step of illuminating the cell of interest comprises using patterned illumination.

12. The method of claim 11, wherein the patterned illumination is performed with a digital micromirror device, galvanometer mirror, acousto-optical beam deflector, or spatial light modulator.

13. The method of claim 1, wherein the step of illuminating the cell of interest comprises illuminating the cell with light having a wavelength of about 360 nm to about 440 nm.

14. The method of claim 1, wherein the step of illuminating the cell of interest comprises illuminating the cell with light having a wavelength of about 440 nm to about 500 nm.

15. The method of claim 1, wherein the step of imaging comprises imaging the cell with a wide-field optical system comprising an objective;

a means of illumination; and

a camera.

16. The method of claim 15, wherein the means of illumination is fluorescent illumination.

18. The method of claim 1, wherein the step of imaging is performed with a high-speed camera.

19. The method of claim 1, wherein the step of imaging is performed using epifluorescensce microscopy, confocal microscopy, differential interference contrast microscopy, phase contrast microscopy, or Raman microscopy.

20. The method of claim 1 further comprising continuing to grow the cell of interest.

21. The method of claim 1 further comprising fixing the cell of interest.

22. The method of claim 1 further comprising removing the cell of interest from the culture.

23. The method of claim 1, wherein the cell of interest is subjected to DNA sequencing, RNA sequencing, or proteomic analysis.

17. The method of 15, wherein the means of illumination utilizes transmitted light.