# US 8,788,213 B2 · Laser mediated sectioning and transfer of cell colonies > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,788,213 B2 - Document ID: US8788213B2 - Status: Granted - Kind: B2 - Application number: 12/686,359 - Filed: 2010-01-12 - Published: 2010-07-22 - Granted: 2014-07-22 - Earliest claimed: 2009-01-12 - Assignees: Intrexon Corporation - Applicants: Gary Bright; Kristi Hohenstein; Anuradha Soundararajan; Manfred Koller - Inventors: Gary Bright; Kristi Hohenstein; Anuradha Soundararajan; Manfred Koller - Web page: https://patentgrep.com/patent/US8788213B2 ## CPC - G01N 1/286: {involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising (microtomes G01N1/06; pulverising in general B02C; mixing in general B01F)} - G01N 15/1433: using image recognition - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The method of sectioning a stem cell colony of claim 1 further comprising adding a dye configured to increase absorbance of the laser energy. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 1) The method of sectioning a stem cell colony of claim 1, further comprising segmenting the imaged cell colony prior to sectioning the colony. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 1) 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. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 1) 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. ### Claim 10 (depends on 1) 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 1) 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. ### Claim 13 (depends on 1) 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. ### Claim 14 (depends on 1) The method of sectioning a stem cell colony of claim 1 further comprising adding a dye configured to increase absorbance of the electromagnetic radiation. ### Claim 15 (depends on 1) 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. ### Claim 16 (depends on 1) The method of sectioning a stem cell colony of claim 1 further comprising removing one or more of the sectioned sub colonies. ### Claim 17 (depends on 16) 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. ### Claim 18 (depends on 16) 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. ### Claim 19 (depends on 16) 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. ### Claim 20 (depends on 16) 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. ### Claim 21 (depends on 16) The method of sectioning a stem cell colony of claim 16 further comprising washing out remaining feeder layer. ### Claim 22 (depends on 1) The method of sectioning a stem cell colony of claim 1, wherein the cell colony comprises living embryonic or induced pluripotent stem cells. ### Claim 23 (depends on 1) The method of sectioning a stem cell colony of claim 1, wherein the cell colony comprises embryonic stem cells or induced pluripotent stem cells.