Cell Colony Picking System

• US 20200131465A1
• Filed: 06/04/2018
• Published: 04/30/2020
• Est. Priority Date: 06/02/2017
• Status: Active Grant

• Current Assignee: Molecular Devices LLC (Danaher Corporation)
• Original Assignee: Molecular Devices LLC (Danaher Corporation)
• Inventors: Floto, Timothy A., Glaser, Alison A., Von Landau, Lee, Phillips, John, Corey, Kyle, Bandhuvula, Padmavathi, Forsyth, Anna Louise
• Granted Patent: US 11,926,813 B2
• Time in Patent Office: Days
• CPC Class Codes: C12M 1/26 Inoculator or sampler
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12M 47/04 Cell isolation or sorting purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04
• CPC Class Codes: G01N 2015/1445 Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography
• CPC Class Codes: G01N 2021/1782 In-depth resolution
• CPC Class Codes: G01N 21/17 Systems in which incident light is modified in accordance with the properties of the material investigated where the material investigated is optically excited causing a change in wavelength of the incident light G01N21/63
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G06T 15/00 3D [Three Dimensional] image rendering
• CPC Class Codes: G06T 15/08 Volume rendering
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/00 Image analysis
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/0016 involving temporal comparison

Abstract

Systems, including methods and apparatus, for growing viable monoclonal colonies, identifying them as monoclonal after their viability has been demonstrated, and picking and placing them into a target environment. The methods may include plating cells at low density in a well, capturing at a first time shortly after plating a series of vertically spaced-apart images of the well showing locations of the cells, capturing at a second, later time images of the same well showing locations of candidate cell colonies, determining a likelihood that the candidate colonies are monoclonal based on information in the first set of images, and, based on the likelihood, picking candidate colonies using a picking head for transfer to a target environment.

Claims

1. A method for selecting one or more monoclonal cell colonies using an apparatus having a digital imaging device, the method comprising:

loading a sample plate onto the apparatus, the sample plate including a well having a plurality of cells held in a semi-solid medium;

capturing, at a first time and using the digital imaging device, a first plurality of vertically spaced-apart images of the well, each image having a different height along a Z axis, such that a volumetric image stack is generated with respect to the well;

identifying, based on one or more images of the well captured at a second time substantially later than the first time, a location of a candidate colony of cells;

determining, based on the volumetric image stack, a measure of likelihood that the candidate colony is monoclonal; and

in response to the determined measure of likelihood, picking the candidate colony using a picking head of the apparatus to isolate at least a portion of the colony.

2. The method of claim 1, further comprising a step of plating the plurality of cells in the semi-solid medium, wherein the step of capturing a first plurality of images is performed less than twenty-four hours after plating.

3. The method of claim 2, wherein the step of capturing a first plurality of images is performed less than six hours after plating.

4. The method of claim 2, further comprising centrifuging the well containing the plated cells between the step of plating the plurality of cells and the step of loading a sample plate onto the apparatus.

5. The method of claim 4, wherein the step of centrifuging the well is performed for at least three minutes and with a centrifugal force of at least 50 times the force of gravity.

6. The method of claim 5, wherein the step of centrifuging the well is performed for about five minutes with a centrifugal force of about 100 times the force of gravity.

7. The method of claim 4, further comprising a step of adding additional semi-solid medium to the well after the step of centrifuging.

8. The method of claim 1, further comprising a step of covering the well with an air-permeable seal before the step of loading the plate.

9. The method of claim 1, wherein a depth of the semi-solid medium in the well is no more than approximately one millimeter.

10. The method of claim 1 wherein respective Z-coordinates of sequential images in the volumetric image stack differ by at least approximately 20 micrometers.

11. The method of claim 10, wherein the respective Z-coordinates of sequential images in the volumetric image stack differ by less than approximately 50 micrometers.

12. The method of claim 11, wherein the respective Z-coordinates of sequential images in the volumetric image stack differ by between 25 micrometers and 45 micrometers.

13. The method of claim 1, wherein the volumetric image stack spans an entire depth of the semi-solid medium in the well.

14. The method of claim 1, wherein the first plurality of images are collected before a majority of the plurality of cells have divided.

15. The method of claim 1, wherein the first plurality of images are collected within approximately six hours after the semi-solid medium has gelled.

16. The method of claim 1, wherein a resolution of the first plurality of images allows identification of single cells.

17. The apparatus of claim 16, wherein an image of a single cell occupies at least about four pixels of the digital imaging device.

18. The method of claim 1, wherein the well has a transparent base, and wherein the digital imaging device has an objective disposed below the base.

19. The method of claim 18, wherein a magnification of the objective is between 2×

and 7×

.

20. The method of claim 18, wherein a numerical aperture of the objective is between 0.1 and 0.4.

21. The method of claim 18, wherein the objective is a dry objective.

22. The method of claim 1, wherein each image of the first plurality of images comprises a combined plurality of smaller images all captured at the same height along the Z-axis.

23. The method of claim 22, wherein the plurality of smaller images are combined based on respective known positions of the digital imaging device with respect to a horizontal plane.

24. The method of claim 1, further comprising:

mapping a plurality of cell locations based on the volumetric image stack;

identifying, from the plurality of cell locations, single cells that are spaced from all other cells by at least a selected first distance; and

limiting potential locations of the candidate colony of cells to locations corresponding to the identified single cells.

25. The method of claim 1, the volumetric image stack being a first volumetric image stack, further comprising:

capturing, before the second time and using the digital imaging device, a second plurality of vertically spaced-apart images of the well, such that a second volumetric image stack is generated with respect to the well; and

comparing the second volumetric image stack to the first volumetric image stack to determine movement of the cells in the semi-solid medium.

26. The method of claim 1, wherein the digital imaging device used to capture the first plurality of vertically spaced-apart images of the well is a first digital imaging device, and the one or more images of the well are captured at the second time using a second digital imaging device.

27. The method of claim 26, wherein the second digital imaging device is disposed above the well.

28. The method of claim 1, wherein the first time and the second time differ by at least ten days.

29. The method of claim 1, wherein the step of identifying a location of a candidate colony of cells further comprises at least one criterion other than monoclonality.

30. The method of claim 29, wherein the at least one criterion is selected from the group consisting of a shape, a density, and a size of the colony.

31. The method of claim 29, wherein the at least one criterion is based at least in part on a proximity of the candidate colony to any other colonies of cells.

32. The method of claim 29, wherein the at least one criterion includes an intensity of fluorescence from the candidate colony.

33. The method of claim 1, wherein the step of determining a measure of likelihood that the candidate colony is monoclonal comprises a step of observing that no more than one cell was found in the volumetric image stack within a selected distance of the location of the candidate colony.

34. The method of claim 33, wherein the distance is no more than about 50 μ

m.

35. The method of claim 1, wherein the step of determining a measure of likelihood that the candidate colony is monoclonal comprises observing how many cell location probability cones intersect a hemispherical volume defined by the selected distance of the candidate colony.

36. The method of claim 1, wherein the step of determining a measuring of likelihood that the candidate colony is monoclonal comprises a step of comparing the location of the candidate colony to a corresponding location in the volumetric image stack.

37. The method of claim 36, wherein the step of comparing the location of the candidate colony to a corresponding location in the volumetric image stack comprises a step of evaluating respective distances between the candidate colony and one or more individual cells locatable in the volumetric image stack.

38. The method of claim 1, further comprising a step of relocating at least a portion of a colony to a dispensing container, after the step of picking.

39. The method of claim 1, further comprising repeating the steps of capturing, identifying, determining, and picking on a second well of the sample plate.

40. An apparatus for picking cell colonies, comprising:

an apparatus bed configured to receive a sample container, wherein the sample container comprises a transparent base supporting a volume of semi-solid medium in which a plurality of cells are present;

a digital camera having an objective disposed below the apparatus bed, such that digital images of the cells in the semi-solid medium are capturable through the transparent base of the sample container;

a Z-axis positioner coupled to the objective and configured to position the objective at a selectable vertical height;

a picking head of the apparatus to isolate at least a portion of the colony; and

a controller coupled to the digital camera, the Z-axis positioner, and picking head, the controller having a processor configured to execute a stored set of instructions (i) to capture a plurality of vertically spaced-apart images of the volume of semi-solid medium, each image taken through the transparent base and having a different height along the Z axis, such that a volumetric image stack is generated, and (ii) to position the picking head to isolate at least a portion of the colony.

41. The apparatus of claim 40, further comprising an X-Y positioner coupled to the objective and the controller and configured to position the objective at a selectable horizontal position.

42. The apparatus of claim 40, wherein the plurality of vertically spaced-apart images are captured at a first time, and wherein the processor is further configured to execute a stored set of instructions to capture one or more images of the volume of semi-solid medium at a second time substantially later than the first time.

43. The apparatus of claim 40, wherein the image of a single cell occupies at least about four pixels of the digital camera.