Optical selection of cells

Patent metadata

Field	Value
Publication number	US-2016208308-A1
Application number	US-201614995716-A
Country	US
Kind code	A1
Filing date	Jan 14, 2016
Priority date	Jan 15, 2015
Publication date	Jul 21, 2016
Grant date	—

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.

First claim

1 . A photochemical crosslinker comprising a fluorescent dye and a radical generator.

2 . The photochemical crosslinker of claim 1 , wherein the photochemical crosslinker is capable of cross-linking a cell to a surface.

3 . The photochemical crosslinker of claim 2 , wherein the cell is a living cell.

4 - 9 . (canceled)

10 . The photochemical crosslinker of claim 1 , wherein the photochemical crosslinker is not cell-permeable.

11 . The photochemical crosslinker of claim 1 , wherein the radical generator is derived from a nitrene radical generator or a carbene radical generator.

12 - 14 . (canceled)

15 . 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 of claim 1 to the culture of cells; and d) illuminating the cell of interest with light, whereby the cell of interest becomes attached to the culture dish.

16 . The method of claim 15 further comprising washing away cells that are not attached to the culture dish through the crosslinker.

17 . The method of claim 15 further comprising isolating the cell of interest from the culture dish.

18 . The method of claim 15 , wherein the culture dish is coated with cell adhesion proteins.

19 . The method of claim 18 , wherein the cell adhesion protein is an extracellular matrix protein.

20 . The method of claim 19 , 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.

21 - 25 . (canceled)

26 . The method of claim 15 , wherein the step of illuminating the cell of interest comprises using patterned illumination.

27 . (canceled)

28 . The method of claim 15 , 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.

29 . The method of claim 15 , 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.

30 . The method of claim 16 , wherein the step of washing away cells comprises incubating the culture of cells with an enzyme.

31 . The method of claim 30 , wherein the enzyme is a proteolytic enzyme.

32 - 37 . (canceled)

38 . A photocleavable crosslinker comprising a photocleavable linker linking two electrophilic groups to each other.

39 - 44 . (canceled)

45 . A method of selecting cells with features of interest comprising:
a) providing a culture of cells in a culture dish; b) attaching the cells to the surface of the culture dish and to their contacting neighbors using a photocleavable crosslinker of claim 38 ; c) imaging the cells to identify a cell of interest; d) adding a protease to the culture of cells; e) illuminating the cell of interest with light, whereby the cell of interest is released from the culture dish.

46 . (canceled)

47 . The method of claim 45 , wherein the culture dish is coated with cell adhesion proteins.

48 - 59 . (canceled)

60 . The method of claim 45 further comprising aspirating the released cell of interest.

61 - 66 . (canceled)

Assignees

• Harvard College

Inventors

• Cohen Adam E
• Chien Miao-Ping

Classifications

• G01N33/582

  with fluorescent label · CPC title

• C12Q1/24 Primary

  Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms · CPC title

• C07D495/04

  Ortho-condensed systems · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• C07D207/46

  with hetero atoms directly attached to the ring nitrogen atom · CPC title