Method for rapid accurate dispensing, visualization and analysis of single cells

• US 10,641,772 B2
• Filed: 02/20/2016
• Issued: 05/05/2020
• Est. Priority Date: 02/20/2015
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Mir, Alain-Albert, Schaal, Thomas D., Lin, Chun-Wah, Nasarabadi, Shanavaz Loharasp D., Dunne, Jude, Srinivasan, Maithreyan, Espinoza, Patricio
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/049,056
• Publication Number:
  US 20160245813A1
• Time in Patent Office: 1,536 Days
• Field of Search: 506 9
• 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 with a liquid dispensing system a dispense volume of a cell suspension into each of at least 75 wells in a multi-well device so that each of the at least 75 wells contains one cell, zero cells or more than one cell and at least some of the wells contain zero cells, wherein said cell suspension comprises cells present at a concentration such that, on average, one cell is present in said dispense volume;

b) capturing a digital image of at least some of the at least 75 wells using an image capturing system;

c) generating a dispense map from the digital image and storing the dispense map to a computer memory, wherein the dispense map provides instructions to the liquid dispensing system to only dispense a reagent into wells containing only one cell; and

d) dispensing, with the liquid dispensing system, the reagent into wells containing only one cell based on the generated dispense map stored in the computer memory.

2. The method of claim 1, wherein said at least 75 wells is at least 150 wells.

3. The method of claim 1, wherein said at least 75 wells is at least 2500 wells.

4. The method of claim 1, wherein the cell suspension comprises labeled cells comprising at least a first detectable label.

5. The method of claim 4, further comprising determining which, if any, of said imaged wells contain a cell labeled with the first detectable label.

6. The method of claim 4, wherein the cell suspension comprises labeled cells comprising at least a first detectable label and a second detectable label.

7. The method of claim 6, further comprising determining which, if any, of said imaged wells contain a cell labeled with the first detectable label and the second detectable label.

8. The method of claim 6, wherein the cell suspension is labeled with the first and second detectable labels prior to the dispensing.

9. The method according to claim 6, wherein the first detectable label is fluorescent at a first wavelength and the second detectable label is fluorescent at a second wavelength.

10. The method according to claim 6, wherein the second detectable label is a cell viability stain or a detectable antibody.

11. The method according to claim 10, wherein the cell viability stain is propidium iodide.

12. The method according to claim 10, wherein the dispense map further identifies specific wells for subsequent dispenses based on the number of viable cell(s) present in each of the imaged wells.

13. The method of claim 4, wherein the cell suspension is labeled with the at least first detectable label prior to the dispensing.

14. The method according to claim 4, wherein the first detectable label is a cellular label.

15. The method according to claim 14, wherein the cellular label comprises Hoechst or a detectable antibody.

16. The method of claim 1, wherein said image capturing system comprises a camera linked to a magnifying lens.

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

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

i) a first list of which wells contain zero cells, ii) a second list of which wells contain one cell; and

iii) a third list of wells that contain more than one cell.

19. The method of claim 18, wherein said image analysis software generates instructions for said liquid dispensing system to dispense a dispense volume of the reagent into each of the wells on said second list.

20. The method according to claim 1, further comprising labeling the cells of the cell suspension with a first detectable label, a second detectable label or both after the dispensing and before the capturing.

21. The method according to claim 1, wherein the reagent is selected from the group consisting of:

a cell culture component, a cell differentiation reagent, a cell surface marker, an antibody, a nanomaterial, a cell lysis reagent, a drug, a nucleic acid, a lipid, a carbohydrate and a protein and combinations thereof.

22. A system comprising:

a) a multi-well device comprising at least 75 wells;

b) a liquid dispensing system that dispenses a dispense volume of a cell suspension into each of said at least 75 wells so that each of the at least 75 wells contains one cell, zero cells or more than one cell and at least some of the wells contain zero cells;

c) an image capturing system configured to capture an image of at least some of the at least 75 wells; and

d) a dispense map file based on the captured image that provides instructions to said liquid dispensing system to only dispense a reagent into wells containing only one cell.

23. The system of claim 22, wherein the image capturing system further comprises a computer comprising a computer processor, computer memory, and image analysis software configured to generate the dispense map file from the captured image.

24. A system comprising:

a) a multi-well device comprising at least 75 wells;

b) a liquid dispensing system that dispenses a dispense volume of a cell suspension into each of said at least 75 wells so that each of the at least 75 wells contains one cell, zero cells or more than one cell and at least some of the wells contain zero cells;

c) an image capturing system configured to capture an image of at least some of the at least 75 wells; and

d) a dispense map file based on the captured image that provides instructions to said liquid dispensing system to only dispense a reagent into wells containing only one cell, wherein the cell suspension is prepared from a tissue.

25. The system of claim 24, wherein the tissue comprises tumor tissue.

26. A system comprising:

a) a multi-well device comprising at least 75 wells;

b) a cell suspension comprising cells present at a concentration such that, on average, one cell is present in a dispense volume;

c) a liquid dispensing system that dispenses said dispense volume of said cell suspension into each of said at least 75 wells so that each of the at least 75 wells contains one cell, zero cells or more than one cell and at least some of the wells contain zero cells;

d) an image capturing system configured to capture an image of at least some of the at least 75 wells; and

e) a dispense map file based on the captured image that provides instructions to said liquid dispensing system to only dispense a reagent into wells containing only one cell, wherein the cells of the cell suspension are labeled with a first detectable label, a second detectable label, or both first and second detectable labels.