Laser mediated sectioning and transfer of cell colonies
View Patent ↗Disclosed herein are methods and devices for sectioning cell colonies. Also disclosed are methods of purifying cell colonies. A method of sectioning cell colonies can include providing a cell colony on a culture plate comprising a known thickness; positioning a bottom of the culture plate using automated focus technology; and sectioning the cell colony into one or more pieces using a pattern of laser cutting lines. Devices for performing the method are also disclosed.
1. A method of sectioning a stem cell colony, comprising:
providing a cell culture comprising at least one living stem cell colony on a culture surface;
imaging the surface;
selecting a stem cell colony for sectioning that exhibits an undifferentiated phenotype for the stem cell colony, which phenotype is shown in the image;
identifying at least a portion of the edge of the stem cell colony in the image;
applying electromagnetic radiation to the identified edge of the stem cell colony to separate the colony from rest of the culture; and
defining a grid pattern of cutting lines which grid pattern defines a plurality of sub colonies of generally uniform size from the selected stem cell colony; and
applying electromagnetic energy in the grid pattern of the cutting lines at the culture surface so as to section the living stem cell colony into the plurality of sub colonies of generally uniform size,
wherein the applying electromagnetic radiation in the grid pattern comprises one or more of: the use of a series of laser pulses which may be emitted using a range of laser energies from about 1 to about 50 μJ per pulse; the use of a series of laser pulses which may be emitted using a range of laser spot radii from about 1 to about 20 μm; and the use of a series of 532 nm laser pulses which may be emitted using 1 to 5 laser pulses.
2. The method of sectioning a stem cell colony of claim 1 further comprising adding a dye configured to increase absorbance of the laser energy.
3. The method of sectioning a stem cell colony of claim 1 , wherein the pattern of electromagnetic cutting lines is limited to within the boundaries of the cell colony.
4. The method of sectioning a stem cell colony of claim 1 , further comprising segmenting the imaged cell colony prior to sectioning the colony.
5. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises the use of a series of laser pulses which may be emitted using a range of laser energies from about 1 to about 50 μJ per pulse.
6. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises use of a series of laser pulses which may be emitted using a range of laser spot radii from about 1 to about 20 μm.
7. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises use of a series of 532 nm laser pulses which may be emitted using 1 to 5 laser pulses.
8. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises use of a series of approximately 532 nm laser pulses which may be emitted using between 1 and 5 laser repeats.
9. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises a pattern of laser cutting lines with pulse spacing between about 5 to about 50 μm.
10. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises a pattern of laser grids with grid spacing between about 5 to about 100 μm.
11. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises a square pattern of laser cutting lines positioned about 20 to about 2000 μm.
12. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises a square pattern of laser cutting lines positioned about 20 to about 300 μm apart for enzyme-free removal of sections.
13. The method of sectioning a stem cell colony of claim 1 , wherein applying electromagnetic radiation in the grid pattern comprises a square pattern of laser cutting lines positioned about 250 to about 2000 μm apart for use in differentiation of stem cells into mature specialized cell types.
14. The method of sectioning a stem cell colony of claim 1 further comprising adding a dye configured to increase absorbance of the electromagnetic radiation.
15. The method of sectioning a stem cell colony of claim 1 further comprising applying electromagnetic radiation to an edge of the cell colony to isolate the living stem cell colony from rest of the culture.
16. The method of sectioning a stem cell colony of claim 1 further comprising removing one or more of the sectioned sub colonies.
17. The method of sectioning a stem cell colony of claim 16 , wherein removing one or more of the sectioned sub colonies comprises dislodging the one or more sectioned sub colonies into suspension within the fluid of a first culture vessel.
18. The method of sectioning a stem cell colony of claim 16 , wherein removing the sectioned one or more sectioned sub colonies comprises addition of chemicals, such as enzymes, prior to dislodging the one or more sectioned sub colonies into suspension within the fluid of the first culture vessel.
19. The method of sectioning a stem cell colony of claim 16 further comprising transferring the one or more sectioned sub colonies to a second culture vessel.
20. The method of sectioning a stem cell colony of claim 16 , wherein removing the sectioned one or more sectioned sub colonies comprises a fluid pipetting technique.
21. The method of sectioning a stem cell colony of claim 16 further comprising washing out remaining feeder layer.
22. The method of sectioning a stem cell colony of claim 1 , wherein the cell colony comprises living embryonic or induced pluripotent stem cells.
23. The method of sectioning a stem cell colony of claim 1 , wherein the cell colony comprises embryonic stem cells or induced pluripotent stem cells.