HIGH DENSITY DEPOSITION FOR ARRAY PRODUCTION

• US 20170307502A1
• Filed: 04/19/2017
• Published: 10/26/2017
• Est. Priority Date: 04/22/2016
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Mason, Clark, Petersen, Timothy W., Azersky, Vladimir, Wolf, Christopher J.
• Granted Patent: US 11,408,812 B2
• Time in Patent Office: Days
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• 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 3/0241 Drop counters; Drop formers making arrays for combinatorial libraries B01J19/0046; automation of dispensing for analysis G01N35/10
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• 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 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1415 Control of particle position

Abstract

Disclosed herein are methods for producing high density cellular arrays. In some embodiments, the methods comprise: providing a sample comprising a plurality of cells; and introducing the plurality of cells in the sample into microwells of a microwell array to produce a cellular array, wherein the microwell array comprises 500 or more microwells per inch², and wherein 25% or more of the microwells of the cellular array comprise a single cell. The disclosed methods can be used for producing a high density synthetic particle array and a high density reagent array.

Claims

1. A method of producing a cellular array, comprising:

providing a sample comprising a plurality of cells; and

introducing the plurality of cells in the sample into microwells of a microwell array to produce the cellular array, wherein the microwell array comprises 500 or more microwells per inch², and wherein 25% or more of the microwells of the cellular array comprise a single cell.

2. The method of claim 1,

wherein introducing the plurality of cells in the sample into the microwells of the microwell array comprises introducing the plurality of cells in the sample into the microwells of the microwell array at a plurality of first desired locations, and

wherein introducing the plurality of cells in the sample into the microwells of the microwell array at the plurality of first desired locations comprises introducing a cell of interest from the plurality of cells at one of the plurality of first desired locations.

3. The method of claim 1, wherein each of the microwells of the microwell array has a volume no more than 1000 nanoliters, wherein each of the microwells of the microwell array has a diameter no more than 1000 micrometers, and wherein at least two of the microwells of the microwell array are separated from each other by no more than 200 micrometers.

4. The method of claim 1, wherein introducing the plurality of cells into the microwells of the microwell array comprises flow cytometrically depositing the plurality of cells into the microwells of the microwell array.

5. The method of claim 1, further comprising introducing a reagent into one or more of the microwells of the cellular array.

6. The method of claim 5, wherein the reagent comprises one or more synthetic particles, and wherein the one or more synthetic particles comprise magnetic beads attached with oligonucleotide barcodes.

7. A cellular array produced by the method of claim 1.

8. A method of aligning a first alignment microwell array for producing a sample microwell array, comprising:

(a) determining a first parameter using a first imaging sensor and a second parameter using a second imaging sensor, wherein the first imaging sensor and the second imaging sensor are approximately orthogonal to each other and are located between a waste receiving vessel and a first alignment microwell array;

(b) providing a desired location on the first alignment microwell array in an x direction and a y direction;

(c) depositing a first alignment droplet into a microwell of the first alignment microwell array based on the first parameter and the second parameter, wherein the first parameter is related to the desired location in the x direction, and wherein the second parameter is related to the desired location in the y direction;

(d) determining the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location;

(e) if the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location is greater than the predetermined threshold value;

adjusting the first parameter and the second parameter based on the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location; and

repeating steps (b)-(d).

9. The method of claim 8, wherein determining the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location comprises imaging the first alignment microwell array and the first alignment droplet using the first imaging sensor and imaging the first alignment array and the first alignment droplet using the second imaging sensor.

10. The method of claim 9, wherein adjusting the first parameter and the second parameter based on the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location comprises determining the first alignment droplet'"'"'s location on the first alignment microwell array based on the imaging of the first alignment microwell array and the first alignment droplet by the first imaging sensor and the imaging of the first alignment microwell and the first alignment droplet by the second imaging sensor.

11. The method of claim 8, wherein depositing the first alignment droplet into the microwell of the first alignment microwell array comprises:

depositing the first alignment droplet into an alignment device of the first alignment microwell array, and wherein determining the distance between the first alignment droplet'"'"'s location on the first alignment microwell array and the desired location comprises imaging the alignment device and the first alignment droplet on the alignment device using the first imaging sensor and imaging the alignment device and the first alignment droplet on the alignment device using the second imaging sensor.

12. The method of claim 11, wherein the alignment device comprises a plurality of alignment regions, wherein two alignment regions of the plurality of alignment regions comprise approximately equal areas, and wherein the plurality of alignment regions comprises a plurality of concentric rings.

13. The method of claim 8, wherein the first parameter is related to droplet charge, or wherein the second parameter is related to the first alignment droplet'"'"'s location in the y direction on the first alignment microwell array.

14. The method of claim 8, wherein the predetermined threshold value correlates to the first alignment droplet'"'"'s distance from an edge of the waste receiving vessel.

15. The method of claim 8, further comprising:

introducing a plurality of sample droplets into microwells of the sample microwell array based on the first parameter and the second parameter, wherein the plurality of sample droplets comprises cells, particles attached with oligonucleotide barcodes, or any combination thereof.

16. The method of claim 15, further comprising:

introducing a second alignment droplet into a microwell of a second alignment microwell array before the plurality of sample droplets are introduced into the microwells of the sample array; and

determining the second alignment droplet'"'"'s location on the second alignment microwell array using the second imaging sensor,

wherein introducing the plurality of sample droplets into the microwells of the sample microwell array is further based on the second alignment droplet'"'"'s location on the second alignment array.

17. The method of claim 8, wherein providing the desired location in the x direction on the first alignment microwell array comprises using the first imaging sensor to determine the desired location in the x direction on the first alignment microwell array and providing the desired location in the y direction on the first alignment microwell array comprises using the second imaging sensor to determine the desired location in the y direction on the sample array.

18. A system for producing a cellular array, comprising:

a cell sorting component configured to;

flow cytometrically introduce droplets into microwells of a microwell array to produce a cellular array, wherein the microwell array comprises 500 or more microwells per inch; and

a control component configured to;

receive a desired location in an x direction and a y direction on the microwell array; and

determine a first parameter using a first imaging sensor and a second parameter using a second imaging sensor for introducing the droplet into a microwell of the microwell array, wherein the first imaging sensor and the second imaging sensor are approximately orthogonal to each other and are between a waste receiving vessel and the microwell array, and

wherein the distance between the droplet'"'"'s location and the desired location is within a predetermined threshold.

19. A method of aligning a flow stream, comprising:

(a) receiving a first parameter used to deposit a first alignment droplet to a desired location on a first alignment microwell array;

(b) depositing a second alignment droplet to a second alignment microwell array using the first parameter;

(c) determining a path of the second alignment droplet, from a cell sorting component to the second alignment microwell, in the first detection field using a first imaging sensor, wherein the first imaging sensor is located between the cell sorting component and a waste receiving vessel; and

(d) determining a first measurement between a first position of a path of the first alignment droplet in the first detection field and a corresponding first position of the path of the second droplet in the first detection field.

20. The method of claim 19, comprising:

(e) if the first measurement is greater than a first predetermined threshold;

adjusting the first parameter based on the first measurement; and

repeating steps (b)-(d).

21. The method of claim 19, wherein the first parameter is related to the desired location in the x direction, or wherein the first parameter is related to droplet charge.

22. The method of claim 19, wherein the first measurement comprises a first distance between the first position of the path of the first alignment droplet in the first detection field and the corresponding first position of the path of the second droplet in the first detection field.

23. The method of claim 19,

wherein receiving the first parameter used to deposit the first alignment droplet to the first desired location on the first alignment microwell array comprises:

depositing the first alignment droplet to the desired location on the first alignment microwell array using the first parameter; and

determining the path of the first alignment droplet in the first detection field using the first imaging sensor.

24. The method of claim 23,

wherein depositing the first alignment droplet to the desired location on the first alignment microwell array using the first parameter comprises:

depositing the first alignment droplet to the desired location on the first alignment microwell array using the first parameter and a second parameter, and

wherein depositing the second alignment droplet to the second alignment microwell array using the first parameter comprises;

depositing the second alignment droplet to the second alignment microwell array using the first parameter and the second parameter.

25. The method of claim 24,

wherein the step (b) further comprises:

determining a path of the first alignment droplet in a second detection field, using a second imaging sensor, from the deflection plate to the desired location on the first alignment microwell array, wherein the second imaging sensor is located between the waste receiving vessel and the deflection plate,

wherein the step (c) further comprises;

determining a path of the second alignment droplet in the second detection field using the second imaging sensor;

wherein the step (d) further comprises;

determining a second measurement between a second position of the path of the first alignment droplet in the second detection field and a corresponding second position of the path of the second droplet in the second detection field, and

wherein the step (f) further comprises;

if the second measurement is greater than a second predetermined threshold, adjusting the second parameter based on the second measurement.

26. The method of claim 25,

wherein determining the path of the first alignment droplet in the first detection field comprises capturing a first alignment image of the first alignment droplet in the first detection field, and

wherein determining the path of the second alignment droplet in the first detection field comprises capturing a second alignment image of the second alignment droplet in the first detection field.

27. The method of claim 26,

wherein determining the path of the first alignment droplet in the first detection field comprises determining a first channel, in the first alignment image, along the path of the first alignment droplet in the first detection field, and

wherein the first measurement comprises whether at least a part of the path of the second alignment droplet in the first detection field is within a corresponding first channel in the second alignment image.

28. The method of claim 27, wherein the first channel is represented by two bars in the first alignment image, and wherein the corresponding first channel is represented by corresponding two bars in the second alignment image.

29. The method of claim 23, wherein depositing the first alignment droplet to the desired location of the first alignment microwell array comprises flow cytometrically depositing the first alignment droplet to the desired location of the first alignment microwell array.

30. A system for producing a cellular array, comprising:

a cell sorting component;

non-transitory memory configured to store executable instructions; and

a processor in communication with the cell sorting component and the non-transitory memory, the processor programmed by the executable instructions to;

(a) receive a first parameter used to deposit the first alignment droplet to the desired location on athe first alignment microwell array;

(b) cause the cell sorting component deposit a second alignment droplet to a second alignment microwell array using the first parameter; and

(c) determine a path of the second alignment droplet, from the cell sorting component to the second alignment microwell, in the first detection field using a first imaging sensor, wherein the first imaging sensor is located between the cell sorting component and a waste receiving vessel; and

(d) determine a first measurement between a first position of a path of the first alignment droplet in the first detection field and a corresponding first position of the path of the second droplet in the first detection field.