Automated cell sample enrichment preparation method

• US 7,494,809 B2
• Filed: 02/14/2006
• Issued: 02/24/2009
• Est. Priority Date: 11/09/2004
• Status: Active Grant

• Current Assignee: VisionGate, Inc.
• Original Assignee: VisionGate, Inc.
• Inventors: Patten, Florence W., Nelson, Alan C.
• Primary Examiner(s): KIM, SUN U
• Application Number:
  US11/353,353
• Publication Number:
  US 20060183220A1
• Time in Patent Office: 1,106 Days
• Field of Search: 210/644, 210/645, 210/649, 210/650, 210/651, 210/767, 210/791, 210/797, 210/798, 210/806, 435/325, 435/289.1
• US Class Current: 435/325
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• 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

Abstract

A method for automated cell enrichment preparation where biological cells are injected into a first subchamber and flowed through the first subchamber, through a large pore filter and into a second subchamber so that the large pore filter retains particles large particles in the first subchamber, while the biological cells pass through the first filter into the second subchamber and are retained by a small pore filter. After clearing, the biological cells are stained. A solvent exchange leaves the biological cells in solvent and they are released into a concentration module where a portion of the solvent is removed to form an enriched concentrated cell suspension.

Claims

1. A method for automated cell enrichment preparation using a chamber having opposing ends, where each opposing end is covered by one of a first small pore filter and a second small pore filter, a large pore filter is mounted between the opposing ends to divide the chamber into a first subchamber and a second subchamber, wherein the large pore filter has a pore size large enough to pass biological cells and the small pore filters have a pore size small enough to retain biological cells, the method comprising the steps of:

injecting a biological cell sample including large particles and biological cells into the first subchamber;

creating a flow of the biological cell sample through the first subchamber, through the large pore filter and into the second subchamber so that the biological cells pass through the large pore filter into the second subchamber and are retained by the second small pore filter, and the large particles are retained by the large pore filter;

using a short impulse back through the second subchamber, large pore filter and into the first subchamber to dislodge the large particles from the large pore filter;

removing the large particles from the first subchamber, while the biological cells remain retained by the second small pore filter in the second subchamber;

staining the biological cells by alternately injecting a stain followed by a rinse through the first subchamber and the second subchamber;

exchanging solvent in the first subchamber and the second subchamber, allowing the biological cells to equilibrate after each exchange until the solvent exchange is complete;

coupling the chamber to a concentration module and releasing the biological cells and solvent from the chamber into the concentration module;

removing solvent from the concentration module to form an enriched concentrated cell suspension;

transferring the enriched concentrated cell suspension into a capillary receptacle coupled to receive the enriched concentrated cell suspension from the concentration module; and

blending the enriched concentrated cell suspension in the capillary receptacle with an optical fluid having an index of refraction selected for improving light transmission through the capillary receptacle.

2. The method of claim 1 wherein the small pore filters have a filter pore size in the range of 8 microns to 10 microns.

3. The method of claim 2 wherein the first small pore filter and the second small pore filter comprise nucleopore polycarbonate filters.

4. The method of claim 1 wherein the large pore filter has a filter pore size in the range of 100 microns to 105 microns.

5. The method of claim 4 wherein the large pore filter comprises a filter selected from the group consisting of a nylon monofilament filter and a polypropylene monofilament filter.

6. The method of claim 1 further comprising the steps of uncoupling the capillary receptacle, and capping and mounting the capillary receptacle in a cassette.

7. The method of claim 6 wherein the capillary receptacle comprises:

a cassette housing having a capillary gripper;

a pair of opposing clips on the top for releasably holding a capillary receptacle;

a plurality of access points for robotic extraction of the capillary receptacle;

a plurality of registration points for automatic alignment verification; and

a plurality of grip points for cassette manipulation.

8. The method of claim 1 wherein each of the small pore filters retain biological cells having a width greater than 10 microns.

9. The method of claim 1 wherein the large pore filter retains large particles having a width greater than 100 microns.

10. The method of claim 1 wherein the capillary receptacle comprises circular or rectangular fused silica capillary tubing.

11. The method of claim 1 wherein the chamber is disposable.

12. The method of claim 1 wherein the optical fluid is an optical gel.

13. The method of claim 12 wherein the optical gel is a thixotropic gel.

14. The method of claim 12 wherein the optical gel has an index of refraction that matches the material of the capillary receptacle.