HIGH-EFFICIENCY SINGLE-CELL COLLECTION METHOD

Abstract

This invention provides a high-efficient single-cell collection method using a specially designed collection well and collection pipet tip for particle/cell collection from the collection well. The structures of the collection well and pipet tip eliminate fluidic dead volume in the collection, resulting in all (or most) of the particles/cells can be brought into the collection pipette tip with the flow. The advantages of this invention in cell manipulation include high cell collection efficiency, low cell damage and easy operation procedure.

Claims

1. A high-efficiency single-cell collecting method, comprising: introducing a liquid containing a plurality of single-cell into a collection well and locating the plurality of single-cell at the bottom of the collection well, inserting a collection pipet tip into the collection well, and giving a suction to draw the liquid with the plurality of single-cell into the collection pipet tip, wherein a gap distance of x is created between the pinpoint of the collection pipet tip and the bottom of the collection well while inserting the collection pipet tip into the collection well, and wherein x is in a range of 10-500 μm.

2. The method of claim 1, wherein the collection pipet tip has a structure of at least three arc-shaped protrusions and an outer wall disposed around the pinpoint of the collection pipet tip.

3. The method of claim 2, wherein there is a height gap between the outer wall and the collection pipet tip.

4. The method of claim 1, wherein the collection well has a structure of at least three arc-shaped protrusions.

5. The method of claim 4, wherein the bottom of the collection well is smaller than the opening of the collection well and the walls of the collection well have a curve.

6. The method of claim 4, the collection well and the collection pipet tip have the same number of the arc-shaped protrusion.

7. The method of claim 1, wherein the collection wall and the collection pipet tip are made of polystyrene (PS), polyethylene (PE), poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polydimethylsilozane (PDMS) or liquid silicone rubber (LSR).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] FIG. 1A-1E show the structure of the collection pipet tip and the collection well. FIG. 1A shows the side view of the collection pipet tip. FIG. 1B shows the top view of the collection pipet tip. FIG. 1C exhibits the closer side view of the pinpoint of the collection pipet tip. FIG. 1D is a perspective drawing showing the structure of the collection well. FIG. 1E shows the side view of inserting the collection pipet tip into the collection well.

[0020] FIG. 2 depicts the single cell isolation flow chart.

[0021] FIG. 3A-3C show the efficiency comparison of the single-cell collection in wells with different structures. FIG. 3A exhibits the particle movement traces of 100 μm of the distance between the bottom and tip edge in the flower-shaped well. FIG. 3B shows the particle movement traces of 200 μm of the distance between the bottom and tip edge in the flower-shaped well. FIG. 3C depicts the particle movement traces of 100 μm of the distance between the bottom and tip edge in the general micro-well.

[0022] FIG. 4A-4C show the comparison of the collection efficiency between general micro-wells and the present collection well. FIG. 4A shows the collection results by using the general micro-wells. FIG. 4B shows the collection results by using the flower-shaped wells. FIG. 4C demonstrates the efficiency comparison between the general micro-wells and the flower-shaped wells

DETAILED DESCRIPTION OF THE INVENTION

[0023] Other features and advantages of the present invention will be further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0025] As used herein, the term of “flower-shaped” in the present disclosure is used to describe a structure of four arc-shaped protrusions.

EXAMPLES

[0026] The other characteristics and advantages of the present invention are further illustrated and described in the following examples. The examples described herein are using for illustrations, not for limitations of the invention.

[0027] The practice of the present invention will employ technologies comprising conventional techniques of cell biology and cell culture, which are within the ordinary skills of the art. Such techniques are explained fully in the literature

Example 1

The Structural Design of the Collection Pipet Tip and the Collection Well

[0028] As shown in FIG. 1A-1D, the collection pipet tip 10 is a pipet tip with a structure of four arc-shaped protrusions and an outer wall 101 around the pinpoint 102. And the collection well 11 is a well with a structure of four arc-shaped protrusions.

[0029] As illustrated in FIG. 1C, the collection pipet tip 10 has an outer wall 101 around pinpoint 102 of the collection pipet tip 10, and there is a height gap 103 between the outer wall 101 and the collection pipet tip 10. On the other hand, referring to FIG. 1D, the bottom 111 of the collection well 11 is smaller than the opening 112 of the collection well 11. As shown in FIG. 1E, when the collection pipet tip 10 is inserted into the collection well 11, the collection pipet tip 10 will not touch the bottom of the collection well 11 because of the wall of the collection well 11 having a curve. As a result, there is a gap distance 104 between them to prevent the collection pipet tip 10 from touching the bottom 111 of the collection well 11, wherein the gap distance is in a range of 10-500 μm.

[0030] Therefore, when the liquid containing particles/cells in the collection well 11 is sucked by the collection pipet tip 10, the pinpoint 102 of the collection pipet tip 10 has a certain distance from the bottom 111 of the collection well 11 and the particles/cells in the liquid are guided by the structure to the bottom 111 of the collection well 11 which is also under the projection of the pinpoint 102 of the collection pipet tip 10.

[0031] By these three-dimensional structure design of the collection well 11 and the collection pipet tip 10, the influence of the boundary effect on the fluid in the collection well 11 is reduced, thereby enabling all (or most) of the particles/cells in the collection well 11 to be driven by the fluid and collected inside of the collection pipet tip 10. The shear stress caused by fluid suction is reduced, which also lowers the chances of damaging the morphology of the single cells.

Example 2

Single Cell Isolation Methodology

[0032] As shown in FIG. 2, the single-cell collection method of the present invention is simple but efficient. First of all, introducing a liquid containing a plurality of desired single-cell or single-particle into the collection well. Second of all, putting the collection pipet tip on a general pipet and inserting the collection pipet tip into the collection well. With the special designed structure of the collection well, the pinpoint of the collection pipet tip has a distance gap from the bottom of the collection well, meaning that the collection pipet tip is not touching the bottom of the collection well and has a space allowing the flow rate of the liquid to change during the suction. Third of all, giving a suction provided by the pipet to draw the liquid into the collection pipet tip. Because of these structures and the gap distance, the flow rate of the liquid in the collection well or the collection pipet tip during the suction operation is increased. And during the suction, the direction of the fluid is from the outer wall side through the bottom to the collection tip, so that all (or most) of the particles/cells in the collection well is brought into the collection pipet tip to achieve the efficient collection.

Example 3

The efficiency of the Collection Well and the Collection Pipet with Floral Shaped Pinpoint

[0033] To deep understanding of how the collection well and the collection pipet tip of the present invention affect particle's/cell's moving traces inside of wells during sipping liquid, a model for particle simulation using COMSOL Multiphysics® Modeling Software is constructed. The particle moving traces of 100 μm and 200 μm distances, which are the gap distance between the edge of the micro-tip and the well's bottom of flower-shaped wells, are compared. As shown in FIG. 3A, 100 μm of the gap in the flower-shaped well can allow all of the particles to move inside the micro-tip within 0.15 seconds. If the gap distance is increased to 200 μm, a few particles retain reside on the well bottom until 0.6 seconds (FIG. 3B). Therefore, the gap distance between tip and well bottom is a critical parameter, which affects particle movement speed. The particles moving traces in the general round-shape vertical sidewall of micro-wells at a gap distance of 100 μm are also simulated. The results demonstrated that some particles remain at the bottom around the sidewall circle of the well since the dead volume affects particle's moving speed (FIG. 3C).

[0034] These results suggest that the well with arc-shaped protrusions has a better efficiency of single-cell collection than the general round-shape well.

Example 4

The Comparison of the Collection Efficiency Between General Micro-Wells and the Present Collection Well

[0035] General micro-well present dead volume near the sidewall and bottom, which is affected by the boundary effect to decrease the flow rate, the area is unable to push particles during absorbing liquid so that particles residue inside a well. Designed collection-wells with flower-shape and the curves of the sidewall, this allows guiding the streamline of flow in the well during liquid absorption to eliminate dead volume. The schematic diagram shows the side view of micro-wells, which retain particle residue (FIG. 4A), but flower-shaped well, which allows guiding the flow streamline in the well during liquid absorption to eliminate dead volume in (FIG. 4B). The particle collection-efficiency of the flower-shaped well is achieving to 100%, which is significantly higher than micro-well 45% (FIG. 4C).

[0036] The above comparisons show that the flower-shaped well with a certain curve near the bottom of the well can guide the flow streamline of the liquid with particles to be well absorbed by the pipet tip and decrease the residue left inside the well.