Manipulation of cells on a droplet actuator

• US 8,716,015 B2
• Filed: 12/15/2008
• Issued: 05/06/2014
• Est. Priority Date: 04/18/2006
• Status: Expired due to Fees

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pollack, Michael G., Pamula, Vamsee K., Eckhardt, Allen E.
• Primary Examiner(s): Tate, Chris R
• Assistant Examiner(s): FIEBIG, RUSSELL G
• Application Number:
  US12/334,575
• Publication Number:
  US 20090155902A1
• Time in Patent Office: 1,968 Days
• Field of Search: None
• US Class Current: 435/325
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: G01N 15/10 Investigating individual particles
• CPC Class Codes: G01N 2015/019 Biological contaminants; Fouling
• CPC Class Codes: G01N 2015/1028 Sorting particles
• CPC Class Codes: G01N 2035/00564 Handling or washing solid phase elements, e.g. beads
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• CPC Class Codes: G01N 35/10 Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices G01N35/0099 takes precedence
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

A method of inoculating a culture medium including providing a droplet including a single cell type on a droplet actuator and inoculating a culture medium with the droplet. A method of providing a metabolically useful substance to a cell culture, including providing a droplet actuator including a cell culture droplet loaded thereon, the sample droplet including cells and a cell culture medium, and a second droplet comprising a metabolically useful substance. The method also includes conducting one or more droplet operations to combine the cell culture droplet with the second droplet on the droplet actuator. Related methods, droplet actuators, and systems are also provided.

Claims

1. A method of inoculating and maintaining a culture medium, the method comprising:

(a) providing a droplet actuator comprising a sample droplet loaded thereon, wherein the sample droplet is substantially surrounded by an oil filler fluid and comprises cells of multiple cell types;

(b) dispensing by electrowetting a sub-droplet from the sample droplet;

(c) analyzing the sub-droplet to determine whether the sub-droplet comprises a single cell type;

(d) repeating steps (b) and (c) until one or more sub-droplets each comprising a single cell type is identified; and

(e) inoculating a culture medium droplet on the droplet actuator by executing electrowetting-mediated droplet operations with a sub-droplet of (c) or (d) comprising a single cell type to bring the sub-droplet into contact with the culture medium droplet to form an inoculated droplet; and

(f) maintaining the inoculated droplet of (e) at a temperature suitable for causing the cells to grow in the inoculated droplet surrounded by the oil on the droplet actuator, and thereby causing the cells to grow.

2. The method of claim 1 wherein the cells comprise cells bound to beads.

3. The method of claim 1 wherein the cells are labeled.

4. The method of claim 3 wherein the cells are labeled with a labeled antibody.

5. The method of claim 1 further comprising combining the culture medium droplet inoculated with the single cell type with a droplet comprising a substance selected from the group consisting of:

beads, particles, microparticles, nanoparticles, drugs, proteins, peptides, antigens, toxins, and antibodies.

6. The method of claim 1 further comprising transporting the culture medium droplet on the droplet actuator.

7. The method of claim 1 wherein the analyzing step comprises conducting an assay comprising droplet-based steps, wherein the assay is mediated by electrowetting electrodes.

8. The method of claim 1 further comprising:

(a) splitting a dispensed sub-droplet of 1(c) comprising multiple cell type to yield two or more daughter droplets; and

(b) analyzing one or more of the daughter droplets for the presence of a single particle.

9. The method of claim 1 further comprising:

(a) combining a dispensed sub-droplet of 1(c) comprising multiple cell type with a buffer droplet;

(b) splitting the dispensed sub-droplet comprising multiple cell type to yield two or more daughter droplets; and

(c) analyzing one or more of the daughter droplets for the presence of a single particle.

10. The method of claim 1 wherein a dispensed sub-droplet of 1(c) lacking cells is transported back to a suspension of cells.

11. The method of claim 1 wherein a dispensed sub-droplet of 1(c) lacking cells is transported into a waste reservoir.

12. The method of claim 1 wherein dispensed sub-droplet sorting decisions are based on a signal detected from the droplet.

13. The method of claim 1 further comprising, after step (f), conducting a droplet based washing protocol to purify the cells of a single cell type.

14. The method of claim 13 wherein the washing protocol comprises a droplet-based merge-and-split protocol.