METHOD FOR RAPID ACCURATE DISPENSING, VISUALIZATION AND ANALYSIS OF SINGLE CELLS

• US 20160245813A1
• Filed: 02/20/2016
• Published: 08/25/2016
• Est. Priority Date: 02/20/2015
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: WaferGen, Inc.
• Inventors: Mir, Alain-Albert, Lin, Chun-Wah, Nasarabadi, Shanavaz Loharasp D., Schaal, Thomas D., Dunne, Jude, Srinivasan, Maithreyan, Espinoza, Patricio
• Granted Patent: US 10,641,772 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/82 Translation products from oncogenes
• CPC Class Codes: G01N 33/57415 of breast
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells
• 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 2207/30242 Counting objects in image
• CPC Class Codes: G06T 7/0012 Biomedical image inspection

Abstract

The present disclosure provides methods, device, assemblies, and systems for dispensing and visualizing single cells. For example, provided herein are systems and methods for dispensing a dispense volume into a plurality of wells of a multi-well device, where, on average, a pre-determined number of cells (e.g., 1-20) are present in the dispense volume, and determining, via a cellular label, the number of cells present in each of the plurality of wells. Such dispensing and cell detection may be repeated a number of times with respect to wells identified as having less than the pre-determined number of cells in order increase the number wells in the multi-well device containing the desired number (e.g., a single cell).

Claims

1. A method comprising:

a) dispensing a dispense volume of a cell suspension into each of a plurality of wells in a multi-well device,

wherein said cell suspension comprises cells present in said cell suspension at a concentration such that, on average, X cell(s) is/are present in said dispense volume, and

wherein said dispensing is performed with a liquid dispensing system;

b) labeling at least a portion of said cells with a first detectable label before and/or after said dispensing; and

c) determining the number of cell(s) present in each of at least some of said plurality of wells.

2. The method of claim 1, wherein X is between 1 and 20 cells.

3. The method of claim 1, wherein X is one.

4. The method of claim 1, wherein said number of cell(s) in at least one of said plurality of wells is determined to be zero.

5. The method of claim 1, wherein said number of cells in at least one of said plurality of wells is between zero and forty.

6. The method of claim 1, further comprising:

d) dispensing a dispense volume of said cell suspension into at least some of said wells determined to have less than X cells.

7. The method of claim 6, further comprising:

e) dispensing a first additional volume which is equal to said dispense volume, but is free of cells, into at least some of said wells determined to have X cells or more than X cells.

8. The method of claim 6, further comprising:

e) determining the number of cell(s) present in each of the wells previously determined to have less than X cells.

9. The method of claim 8, further comprising:

f) dispensing a dispense volume of said cell suspension into wells determined to have less than X cells.

10. The method of claim 9, further comprising:

g) dispensing a second additional volume which is equal to said dispense volume, but is free of cells, into at least some of said wells determined to have X cells or more than X cells.

11. The method of claim 1, further comprising:

labeling at least a portion of said cells with a second detectable label before and/or after said dispensing and/or determining steps.

12. The method of claim 11, further comprising:

determining which, if any, of said plurality of wells contain a cell with said second label.

13. The method of claim 1, wherein said determining comprises visualizing said first detectable label in each of said at least some of said plurality of wells.

14. The method of claim 1, wherein said determining comprising capturing a first image of at least some of said plurality of wells using an image capturing system, wherein said first image indicates the number of cells present in each of the wells in said first image.

15. The method of claim 14, wherein said image capturing system comprises a camera linked to a magnifying lens.

16. The method of claim 15, wherein said image capturing system further comprises a computer, wherein said computer comprises a computer processor, computer memory, and image analysis software.

17. The method of claim 16, wherein said image analysis software is configured to analyze said first image and generate:

i) a first list of which wells contain less than X cells, ii) a second list of which wells contain X cells; and

iii) third list of wells than contain more than X cells.

18. The method of claim 17, wherein said image analysis software generates instructions, for said liquid dispensing system, to dispense a dispense volume into each of said first list of wells.

19. The method of claim 17, further comprising:

d) dispensing, based on said first image, a dispense volume into at least some of said wells with less than X cells.

20. The method of claim 19, further comprising:

e) capturing a second image of at least some of said wells with less than X cells prior to said dispensing, wherein said second image indicates, via said first detectable label, the number of cell(s) present in each of said wells with less than X cells prior to said dispensing.

21. A system comprising:

a) a multi-well device comprising a plurality of wells;

b) a liquid dispensing system configured to dispense a dispense volume into each of said plurality of wells; and

c) at least one component selected from;

i) a cell suspension comprising cells present in said cell suspension at a concentration such that, on average, X cell(s) is/are present in said dispense volume,

ii) a dispense map file that provides instructions to said liquid dispensing system for dispensing liquid into cells in said multi-well device that contain X cells.