Single cell collection method

Abstract

To provide 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.SOLUTION: The structures of a collection well 11 and a pipette tip 10 eliminate fluidic dead volume in collection, resulting in all (or most) of particles / cells can be brought into the collection pipette tip 10 with flow. The advantages of the invention in cell manipulation include high cell collection efficiency, low cell damage, and easy operation procedure.SELECTED DRAWING: Figure 1E

Description

[Technical field]

[0001] The present invention relates to the field of molecular biology, and more specifically to a highly efficient single cell collection method for gently trapping single cells without affecting cell morphology. [Background technology]

[0002] Single-cell analysis can reveal important clues from rare cells, which is essential for understanding complex biological regulation within heterogeneous populations. Recently, advances in cell handling and sequencing technologies have enabled the analysis of single-cell genomes / transcriptomes using commercialized systems, including the 10X Genome System, Cellsearch System, and Fluidigm C1. Such systems can provide high-throughput, single-cell level genome / transcriptome information to discover new biological mechanisms and develop new therapeutic strategies to treat diseases. Essentially, single-cell analysis has been widely used in various biological fields and has the opportunity to be applied in clinical diagnosis, for example, to analyze circulating tumor cells isolated from the peripheral blood of cancer patients. Summary of the Invention [Problem to be solved by the invention]

[0003] However, the size of a single cell is only 10-20 μm, which makes the manipulation of single cells very difficult. The present invention also emphasizes the need for a special method or device for collecting single cells. Current single cell manipulation methods, such as using micromanipulators, flow cytometers, single cell pipettes, and filters, are still limited by low efficiency, high shear stress, high cost, and other problems. For example, by using filters to collect single cells, the cells are susceptible to structural squeezing forces and fluid shear forces. On the other hand, although flow cytometers are very efficient in collecting single cells, the cells are also at risk of being damaged by laser and fluid shear forces, and it has a higher cost. Highly efficient single cell collection methods are urgently needed for single cell genotyping and phenotyping.

[0004] Therefore, the present invention provides a highly efficient single cell collection method using a collection well and a collection pipette tip with a new structure to reduce the influence of boundary effects on the fluid in the collection well, thereby allowing all (or most) of the particles / cells in the collection well to be driven by the fluid and collected in the collection pipette tip. By these means, the objective of low shear stress, fast and effective collection can be achieved. Moreover, the present invention can be operated manually using a common pipette, reducing the cost of purchasing and maintaining the equipment. [Means for solving the problem]

[0005] In one aspect, the present invention provides a highly efficient method for collecting single cells, which includes introducing a liquid containing one or more single cells into a collection well, disposing the one or more single cells at the bottom of the collection well, inserting a collection pipette tip into the collection well, and applying suction to draw the liquid with the one or more single cells into the collection pipette tip. The collection pipette tip has at least three arcuate projections extending from the outer wall of the collection pipette tip, and the collection well has an inner wall extending from the inner wall of the collection well. and is provided to increase the volume of the space surrounded by the inner wall of the collection well. It has a structure of at least three arc-shaped protrusions. The bottom of the collection well is smaller than the top opening of the collection well, and the bottom of the inner wall of the collection well has a curved shape. After inserting the collection pipette tip into the collection well, a gap distance x is generated between the pin tip of the collection pipette tip and the bottom of the collection well, where x is in the range of 10 to 500 μm.

[0006] The method presented in this disclosure provides a new means for capturing or collecting single cells or a small number of cells in a fast and efficient manner. In addition to being highly efficient, this method also has the advantages of high performance and low cost. It causes low cell damage and can be used with a general optical microscope to obtain high content cell images for multidirectional single cell analysis. Also, the present invention does not require high equipment costs because it can be operated manually with a general pipette.

[0007] In one embodiment, the collection pipette tip and collection well are made of polystyrene (PS), polyethylene (PE), polymethylmethacrylate (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), or liquid silicone rubber (LSR).

[0011] In another preferred embodiment, the bottom of the collection well is top Smaller than the opening of the collection well Inside wall Bottom of face is a curve shape has.

[0012] In one embodiment, the collection well Inner wall surface of and collection pipette tip Exterior wall surface have the same number of arc-shaped protrusions.

[0013] In one embodiment, the collection well and collection pipette tip have a flower-shaped structure, four arc-shaped protrusions.

[0014] In one embodiment, the aspiration is performed by a common pipette.

[0015] Single cell analysis is the identification of differences between individual cells in an entire population, helping to understand the heterogeneity and behavior of the population. Analysis of cell populations only considers average attributes, but cannot identify small important variations between individual cells of a sample. These reasons highlight the importance of how to isolate and obtain single cells, and also highlight the value of the present invention, which is easily manipulated and portable.

[0016] Therefore, in another aspect, the present invention can be extended to various applications such as portable chip design, single cell manipulation, immunology, cancer and stem cell research, disease diagnosis and drug development, such as: 1) stem cell / cancer cell heterogeneity analysis, 2) single cell lineage establishment, 3) monoclonal antibody drug development, 4) single cell related research (nucleic acid sequencing, protein analysis and single cell culture), 5) concentration of cell samples / bioparticles, 6) analysis and experiment of very small number of cells (such as circulating tumor cells).

[0017] This patent or application contains at least one drawing executed in color. [Brief description of the drawings]

[0018] [Figure 1A] FIG. 2 is a side view of a collection pipette tip. [Figure 1B] FIG. 2 is a plan view showing the structure of a collection pipette tip. [Figure 1C] FIG. 13 is a close-up side view of the pin tip of a collection pipette tip. [Figure 1D] FIG. 2 is a perspective view showing the structure of a collection well. [Figure 1E] FIG. 13 is a side view of a collection pipette tip inserted into a collection well. [Diagram 2] 1 shows a flow chart of single cell isolation. [Figure 3A] Particle migration traces in a flower-shaped well with a distance of 100 μm between the bottom and the chip edge are shown. [Figure 3B] Particle migration traces in a flower-shaped well with a distance of 200 μm between the bottom and the edge are shown. [Figure 3C] FIG. 1 shows particle movement traces in a typical microwell with a distance of 100 μm between the bottom and edge. [Figure 4A] 4 shows the results of collection using a typical microwell. [Figure 4B] The results of collection using a flower-shaped well are shown. [Figure 4C] A comparison of collection efficiency between a general microwell and a flower-shaped well is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Other features and advantages of the present invention are further illustrated and described in the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] As used herein, the term "flower-shaped" in this disclosure is used to describe a structure of four arcuate projections.

[0022] (Example) Other features and advantages of the present invention are further illustrated and described in the following examples. The examples described herein are used for illustration purposes and are not used to limit the present invention.

[0023] The practice of the present invention employs techniques, including conventional techniques of cell biology and cell culture, which are within the ordinary skill of the art. Such techniques are fully explained in the literature.

[0024] Example 1. Structural design of collection pipette tip and collection well 1A-1D, the collection pipette tip 10 is a pipette tip having a structure of four arcuate protrusions and an outer wall 101 around a pin tip 102. And the collection well 11 is a well having a structure of four arcuate protrusions.

[0025] As shown in FIG. 1C, the collection pipette tip 10 has an outer wall 101 around the pin tip 102 of the collection pipette tip 10, and there is a height gap 103 between the outer wall 101 and the collection pipette tip 10. Meanwhile, 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 pipette tip 10 is inserted into the collection well 11, the collection pipette tip 10 does not contact the bottom 111 of the collection well 11 because the wall of the collection well 11 is curved. As a result, there is a gap distance 104 between them to prevent the collection pipette tip 10 from contacting the bottom 111 of the collection well 11, and the gap distance is in the range of 10 to 500 μm.

[0026] Therefore, when the liquid containing particles / cells in the collection well 11 is aspirated by the collection pipette tip 10, the pin tip 102 of the collection pipette 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 under the protrusion of the pin tip 102 of the collection pipette tip 10.

[0027] These three-dimensional structural designs of the collection well 11 and the collection pipette tip 10 reduce the influence of boundary effects on the fluid in the collection well 11, thereby allowing all (or most) of the particles / cells in the collection well 11 to be driven by the fluid and collected in the collection pipette tip 10. The shear stress caused by fluid suction is reduced, which also reduces the possibility of damaging the morphology of single cells.

[0028] Example 2. Single cell isolation methodology. As shown in FIG. 2, the single cell collection method of the present invention is simple but efficient. First, a liquid containing multiple desired single cells or single particles is introduced into the collection well. Second, a collection pipette tip is attached to a common pipette and the collection pipette tip is inserted into the collection well. With the specially designed structure of the collection well, the pin tip of the collection pipette tip has a gap distance from the bottom of the collection well. This means that the collection pipette tip does not contact the bottom of the collection well and has a space that allows the flow rate of the liquid to change during aspiration. Third, a suction force provided by the pipette is given to draw the liquid into the collection pipette tip. Due to these structures and gap distances, the flow rate of the liquid in the collection well or collection pipette tip during the aspiration action is increased. And during aspiration, the direction of the fluid is from the outer wall side through the bottom toward the collection pipette tip. As a result, all (or most) of the particles / cells in the collection well are brought to the collection pipette tip to achieve efficient collection.

[0029] Example 3. Efficiency of Collection Pipette Tips with Collection Wells and Floral Pin Tips To gain a deeper understanding of how the collection well and collection pipette tip of the present invention affect the migration traces of particles / cells in the wells while sucking up the liquid, a model was built for particle simulation using COMSOL Multiphysics® modeling software. Particle migration traces with a gap distance of 100 μm and 200 μm between the edge of the microtip and the bottom of the well of the flower-shaped well are compared. As shown in Figure 3A, the gap of 100 μm in the flower-shaped well allows all particles to move into the microtip within 0.15 seconds. When the gap distance is increased to 200 μm, a few particles remain at the bottom of the well until 0.6 seconds (Figure 3B). Thus, the gap distance between the tip and the bottom of the well is an important parameter that affects the particle migration speed. The particle migration traces with a gap distance of 100 μm in the vertical sidewall of the typical round shape of the microwell were also simulated. The results demonstrated that some particles remain at the bottom around the sidewall circle of the well because the dead volume affects the migration speed of the particles (Figure 3C).

[0030] These results suggest that the wells with arc-shaped protrusions are more efficient at collecting single cells than general round wells.

[0031] Example 4. Comparison of collection efficiency between a general microwell and the collection well of the present invention. A typical microwell has dead volumes near the sidewalls and bottom, which are affected by boundary effects that reduce the flow rate, and the area cannot push out particles while absorbing liquid, resulting in particles remaining in the well. The designed collection well with flower shape and sidewall curvature can thereby guide the flow streamline in the well during liquid absorption to eliminate the dead volume. The schematic diagram shows a side view of a microwell that retains particle residues in Figure 4A, while in Figure 4B, a side view of a flower-shaped well that allows guiding the flow streamline in the well during liquid absorption to eliminate the dead volume. The particle collection efficiency of the flower-shaped well reaches 100%, which is significantly higher than the 45% of the microwell (Figure 4C).

[0032] The above comparison shows that the flower-shaped well, which has a certain curve near the bottom of the well, can guide the flow line of the particle-containing liquid so that it can be fully absorbed by the pipette tip, reducing the residue left inside the well. [Explanation of symbols]

[0033] 10 Collection Pipette Tips 101 Exterior Wall 102 Pin 103 Height Gap 104 Gap Distance 11 Collection well 111 Bottom

Claims

1. A highly efficient single cell collection method, comprising: introducing a liquid containing one or more single cells into a collection well and disposing said one or more single cells at a bottom of said collection well; Inserting a collection pipette tip into said collection well; applying aspiration to draw the liquid along with the single cell or cells into the collection pipette tip; Including, a collection pipette tip having a structure of at least three arc-shaped protrusions extending from an outer wall surface of the collection pipette tip; a collection well having a structure of at least three arc-shaped protrusions extending to an inner wall surface of the collection well and arranged to increase a volume of a space surrounded by the inner wall of the collection well; a bottom of the collection well is smaller than a top opening of the collection well; a bottom of the inner wall surface of the collection well has a curved shape; and after the collection pipette tip is inserted into the collection well, a gap distance x is generated between the pin tip of the collection pipette tip and the bottom of the collection well, where x is in the range of 10 to 500 μm.

2. The method for collecting a single cell according to claim 1 , wherein the number of the arc-shaped protrusions on the inner wall surface of the collection well and the outer wall surface of the collection pipette tip are the same.

3. 2. The single cell collection method of claim 1, wherein the collection well and the collection pipette tip are made of polystyrene (PS), polyethylene (PE), polymethyl methacrylate (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), or liquid silicone rubber (LSR).

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