WO2018022159A1

WO2018022159A1 - Single-cell pipette assembly comprising single-cell pipette handle and single-cell pipette tip

Info

Publication number: WO2018022159A1
Application number: PCT/US2017/030629
Authority: WO
Inventors: Lidong Qin; Kai Zhang
Original assignee: The Methodist Hospital
Priority date: 2016-07-27
Filing date: 2017-05-02
Publication date: 2018-02-01
Also published as: WO2018022159A9

Abstract

Apparatus for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, the apparatus comprising: a single-cell pipette tip, the single-cell pipette tip comprising: a structure having a distal end; a Y-shaped microchannel formed in the structure, the Y-shaped microchannel comprising a base microchannel, a first branch microchannel and a second branch microchannel, wherein the base microchannel extends to the distal end of the structure, the first branch microchannel is connectable to a pressure channel, and the second branch microchannel is connectable to a pressure channel; and a single-cell trap formed in the structure and in communication with the base microchannel, the single-cell trap being disposed distal to the convergence of the base microchannel with the first branch microchannel and the second branch microchannel.

Description

SINGLE-CELL PIPETTE ASSEMBLY COMPRISING SINGLE-CELL PIPETTE HANDLE AND SINGLE-CELL PIPETTE TIP

Applicant

The Methodist Hospital

Reference To Pending Prior Patent Applications

This patent application claims benefit of pending prior U.S. Patent Application Serial No. 15/114,704, filed 07/27/2016 by The Methodist Hospital and Lidong Qin et al . for SINGLE-CELL PIPETTE ASSEMBLY COMPRISING

SINGLE-CELL PIPETTE HANDLE AND SINGLE-CELL PIPETTE TIP (Attorney's Docket No. METHODI ST-9 PCT US), which patent application claims benefit of prior

International (PCT) Patent Application No.

PCT/US2015/013343, filed 01/28/2015 by The Methodist

Hospital for SINGLE-CELL PIPETTE ASSEMBLY COMPRISING SINGLE-CELL PIPETTE HANDLE AND SINGLE-CELL PIPETTE TIP (Attorney's Docket No. METHODI ST-9 PCT), which patent application claims benefit of prior U.S. Provisional Patent Application Serial No. 61/932,493, filed

01/28/2014 by The Methodist Hospital and Lidong Qin et al. for SINGLE CELL PIPETTE (SCP) AND SINGLE CELL PIPETTE TIP (SCP-TIP) (Attorney's Docket No.

METHODI ST-9 PROV) .

The three (3) above-identified patent

applications are hereby incorporated herein by

reference .

Field Of The Invention

This invention relates to cell manipulation in general, and more particularly to isolating an

individual cell from a group of cells and transferring that isolated cell to a desired location.

Background Of The Invention

Current biological research and clinical cell analysis frequently requires the isolation of an individual cell from a group of cells and the transfer of that isolated cell to a desired location (e.g., common 96- or 384-well plates, cell culture dishes, vials, microscope slides, etc.) . Ideally, such isolation and transfer should be well controlled, highly efficient, operationally simple, fast to implement and inexpensive. However, for a variety of reasons, none of the approaches available to date are completely satisfactory.

Thus, a new approach is needed for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location. Summary Of The Invention

The present invention provides a new approach for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location.

More particularly, the present invention provides a novel single-cell pipette assembly for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location.

In one preferred form of the invention, the single-cell pipette assembly comprises a single-cell pipette handle and a single-cell pipette tip. The single-cell pipette handle comprises a first pressure channel and a second pressure channel. The single- cell pipette tip comprises a Y-shaped microchannel having a base microchannel, a first branch

microchannel and a second branch microchannel. The base microchannel extends to the distal end of the single-cell pipette tip. The first branch

microchannel of the Y-shaped microchannel is connected to the first pressure channel of the single-cell pipette handle. The second branch microchannel of the Y-shaped microchannel is connected to the second pressure channel of the single-cell pipette handle. A single-cell trap is disposed in the base microchannel of the Y-shaped microchannel, distal to the

convergence of the base microchannel with the first branch microchannel and the second branch

microchannel . On account of the foregoing construction, when the base microchannel , first branch microchannel and second branch microchannel are primed with primer solution, and the base microchannel is disposed in a slurry of cells, and negative pressure is applied to the first pressure channel, the slurry of cells is drawn up into the base microchannel and into the first branch microchannel, with a single cell from the slurry being captured in the single-cell trap. Next, the base microchannel is disposed in a wash solution, and negative pressure is applied to the first pressure channel so that the wash solution flushes the slurry of cells out of the base microchannel, with the captured cell remaining in the single-cell trap.

Thereafter, when the single cell captured in the single-cell trap is to be transferred to a desired location, positive pressure is applied to the second pressure channel so that the primer solution is flushed through the second branch microchannel, into the base microchannel and then out the distal end of the single-cell pipette tip, whereby to flush the captured cell out of the single-cell trap, through the base microchannel and then out the distal end of the single-cell pipette tip.

In one preferred form of the present invention, there is provided apparatus for isolating an

individual cell from a group of cells and transferring that isolated cell to a desired location, said

apparatus comprising:

a single-cell pipette tip, said single-cell pipette tip comprising:

a structure having a distal end;

a Y-shaped microchannel formed in said structure, said Y-shaped microchannel comprising a base microchannel, a first branch microchannel and a second branch microchannel, wherein said base

microchannel extends to said distal end of said structure, said first branch microchannel is

connectable to a first pressure channel, said second branch microchannel is connectable to a second

pressure channel, and a single-cell trap is disposed in said base microchannel, distal to the convergence of said base microchannel with said first branch microchannel and said second branch microchannel.

In another preferred form of the present

invention, there is provided a method for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, said method comprising :

providing a single-cell pipette tip, said single- cell pipette tip comprising:

a structure having a distal end;

pressure channel, and a single-cell trap is disposed in said base microchannel, distal to the convergence of said base microchannel with said first branch microchannel and said second branch microchannel;

priming said base microchannel, said first branch microchannel and said second branch microchannel with primer solution;

positioning said base microchannel in a slurry of cells ;

applying negative pressure to said first branch microchannel and said base microchannel so that the slurry of cells is drawn up into said base

microchannel and into said first branch microchannel, with a single cell from the slurry being captured in said single-cell trap;

positioning said base microchannel in a wash solution;

applying negative pressure to said first branch microchannel and said base microchannel so that said wash solution flushes the slurry of cells out of said base microchannel, with the captured cell remaining in said single-cell trap; and thereafter, when the single cell captured in said single-cell trap is to be transferred to a desired location, applying positive pressure to said second pressure channel so that said primer solution is flushed through said second branch microchannel , into said base microchannel and then out said distal end of said single-cell pipette tip, whereby to flush the captured cell out of said single-cell trap, through said base microchannel and then out said distal end of the single-cell pipette tip.

In accordance with the present invention, the successful capture of a cell in the single-cell trap may be confirmed visually, e.g., via microscope.

And in accordance with the present invention, the single-cell pipette tip may be provided with a

geometry to facilitate viewing of the single-cell pipette tip under a microscope.

And in accordance with the present invention, the apparatus may comprise a "dual-channel" Air

Displacement Pipette (ADP) for applying negative and/or positive pressure to the single-cell pipette tip, or the apparatus may comprise a "single-channel" Air Displacement Pipette (ADP) for applying negative and/or positive pressure to the single-cell pipette tip. Where the apparatus comprises a "single-channel"

Air Displacement Pipette (ADP) for applying negative and/or positive pressure to the single-cell pipette tip, a dual-channel adapter may be disposed between the "single-channel" Air Displacement Pipette (ADP) and the single-cell pipette tip.

And in accordance with the present invention, the single-cell trap may comprise a hook structure

disposed in the base microchannel of the single-cell pipette tip, or the single-cell trap may comprise a passageway having an inlet/outlet configuration disposed in a side wall separating two serial lengths of the base microchannel.

In one form of the invention, there is provided apparatus for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, said apparatus comprising:

a single-cell pipette tip, said single-cell pipette tip comprising:

a structure having a distal end;

connectable to a pressure channel, and said second branch microchannel is connectable to a pressure channel; and

a single-cell trap formed in said structure and in communication with said base microchannel, said single-cell trap being disposed distal to the convergence of said base microchannel with said first branch microchannel and said second branch

microchannel .

In another form of the invention, there is provided a method for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, said method comprising: providing a single-cell pipette tip, said single- cell pipette tip comprising:

a structure having a distal end;

a single-cell trap formed in said structure and in communication with said base microchannel, said single-cell trap being disposed distal to the

convergence of said base microchannel with said first branch microchannel and said second branch

microchannel;

priming said base microchannel, said first branch microchannel and said second branch microchannel with primer solution; positioning said base microchannel in a slurry of cells ;

positioning said base microchannel in a wash solution;

applying negative pressure to said first branch microchannel and said base microchannel so that said wash solution flushes the slurry of cells out of said base microchannel, with the captured cell remaining in said single-cell trap; and

thereafter, when the single cell captured in said single-cell trap is to be transferred to a desired location, applying positive pressure to said second branch microchannel so that said primer solution is flushed through said second branch microchannel, into said base microchannel and then out said distal end of said single-cell pipette tip, whereby to flush the captured cell out of said single-cell trap, through said base microchannel and then out said distal end of the single-cell pipette tip.

Brief Description Of The Drawings These and other objects and features of the present invention will be more fully disclosed or rendered obvious by the following detailed description of the preferred embodiments of the invention, which is to be considered together with the accompanying drawings wherein like numbers refer to like parts, and further wherein:

Fig. 1 is a schematic view showing a novel single-cell pipette assembly formed in accordance with the present invention, wherein the single-cell pipette assembly comprises a single-cell pipette handle and a single-cell pipette tip;

Fig. 2 is a schematic view showing the single- cell pipette handle of the novel single-cell pipette assembly of Fig. 1 ;

Fig. 3 is a schematic view showing connection tubes for connecting the single-cell pipette handle of the novel single-cell pipette assembly of Fig. 1 to the single-cell pipette tip of the novel single-cell pipette assembly of Fig. 1 ;

Figs. 4 and 5 are schematic views showing details of the single-cell pipette tip of the novel single- cell pipette assembly of Fig. 1, including the single- cell trap disposed within the single-cell pipette tip;

Figs. 6-8 are schematic views showing further details of the single-cell trap shown in Fig. 5, including showing the single-cell trap holding a captured cell and releasing the captured cell; Figs. 9A-9D are schematic views showing how a slurry of cells may be flowed by the single-cell trap of the single-cell pipette tip, an individual cell captured in the single-cell trap, and the individual cell thereafter released from the single-cell trap;

Fig. 10 is a schematic view showing various ways in which the base microchannel of the single-cell pipette tip may be configured to facilitate capture of a single cell by the single-cell trap;

Fig. 10A is a schematic view showing a simple model of single-cell capture by the single-cell trap;

Fig. 10B is a graph showing single-cell capture efficiency under various fluid-resistance rations Fig. IOC is a graph showing single-cell capture efficiency under various aspiration times and cell concentrations ;

Fig. 10D is a table showing single-cell capture efficiency with various bypass path widths;

Fig. 10E is a table showing single-cell capture efficiency with various bypass path lengths;

Figs. 11-19 are schematic views showing how the novel single-cell pipette assembly of Fig. 1 may be used to isolate an individual cell from a group of cells and transfer that isolated cell to a desired location;

Fig. 19A is a schematic view showing a novel single-cell pipette assembly formed in accordance with the present invention, wherein the single-cell pipette assembly comprises a single-cell pipette handle and a single-cell pipette tip, and further wherein the single-cell pipette tip comprises a flat surface for enabling the single-cell pipette tip to remain

stationary on a surface, e.g., while being viewed using a microscope;

Fig. 19B is a schematic view showing the distal end of the single-cell pipette tip shown in Fig. 19A;

Figs. 20-28 are schematic views similar to those of Figs. 11-19, except showing a modified form of single-cell pipette assembly isolating an individual cell from a group of cells and transferring that isolated cell to a desired location;

Fig. 28A is a schematic view showing another novel form of single-cell pipette assembly, wherein the single-cell pipette tip comprises multiple single- cell traps;

Fig. 29 is a schematic view showing another novel form of single-cell pipette assembly, wherein the single-cell pipette assembly comprises multiple single-cell pipette tips;

Fig. 30 is a schematic view similar to that of Fig. 29, except showing a modified form of single-cell pipette handle;

Fig. 31 is a schematic view showing another novel form of single-cell pipette assembly, wherein the single-cell pipette assembly comprises multiple single-cell pipette tips;

Fig. 32 is a schematic view similar to that of Fig. 31, except showing a modified form of single-cell pipette handle;

Fig. 33 is a schematic view showing another form of single-cell pipette tip formed in accordance with the present invention, wherein the body of the single- cell pipette tip comprises a plurality of Y-shaped microchannels each comprising a single-cell trap, and further wherein the plurality of Y-shaped

microchannels share a common distal end inlet /outlet ;

Fig. 34 is a schematic view showing how a cell captured in a single-cell trap of a Y-shaped

microchannel may be cultured prior to release from the

Y-shaped microchannel;

Figs. 35-37 are schematic views showing a

"single-channel" Air Displacement Pipette (ADP) and a dual-channel adapter for applying negative and/or positive pressure to the single-cell pipette tip;

Fig. 38 is a schematic view showing the single- cell pipette tip of Figs. 35-37 disposed in a Petri dish for microscopic examination; and

Figs. 39-42 are schematic views showing a single- cell trap which comprises a passageway having an inlet/outlet configuration disposed in a side wall separating two serial lengths of the base

microchannel . Detailed Description Of The Preferred Embodiments

More particularly, the present invention

comprises the provision and use of a novel single-cell pipette assembly for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location.

Novel Single-Cell Pipette Assembly In General

In one preferred form of the invention, and looking now at Figs. 1-5, there is provided a single- cell pipette assembly 5. Single-cell pipette assembly 5 comprises a single-cell pipette handle 10 and a single-cell pipette tip 15. Single-cell pipette handle 10 and single-cell pipette tip 15 are

preferably formed as separate elements which are assembled by the user prior to use, although they may also be formed as a single integral member if desired. For purposes of the present invention, single-cell pipette handle 10 and single-cell pipette tip 15 will be discussed in the context of being formed as

separate elements which are assembled by the user prior to use.

Single-cell pipette handle 10 comprises a body 20, a first pressure channel 25 and a second pressure channel 30. In one preferred form of the invention, first pressure channel 25 comprises a first passageway 35 extending through body 20 and communicating with a first connection tube 40; and second pressure channel 30 comprises a second passageway 45 extending through body 20 and communicating with a second connection tube 50. In one preferred form of the invention, a first plunger 55 is movably disposed in first

passageway 35, such that movement of first plunger 55 in first passageway 35 can apply positive or negative pressure to first connection tube 40; and a second plunger 60 is movably disposed in second passageway 45, such that movement of second plunger 60 in second passageway 45 can apply positive or negative pressure to second connection tube 50. It should be

appreciated that, if desired, single-cell pipette handle 10 may comprise a traditional Air Displacement Pipette (ADP) of the sort well known in the art for providing a negative pressure channel (e.g., first pressure channel 25) operated by moving a first mechanism (e.g., first plunger 55), and a positive pressure channel (e.g., second pressure channel 30) operated by moving a second mechanism (e.g., second plunger 60) . It should also be appreciated that first connection tube 40 and second connection tube 50 are preferably detachable from body 20 of single-cell pipette handle 10, such that first connection tube 40 and second connection tube 50 may be discarded after use (or appropriately cleaned and sterilized for subsequent reuse if desired) .

Single-cell pipette tip 15 comprises a body 65 having a Y-shaped microchannel 70 formed therein.

Body 65 has a distal tip 75. Y-shaped microchannel 70 comprises a base microchannel 80, a first branch microchannel 85 and a second branch microchannel 90, with base microchannel 80, first branch microchannel 85 and second branch microchannel 90 converging at a convergence point 95. Base microchannel 80 extends from convergence point 95 to distal tip 75. First branch microchannel 85 extends from convergence point 95 to a first coupling 100, where first branch

microchannel 85 connects to first connection tube 40. Second branch microchannel 90 extends from convergence point 95 to a second coupling 105, where second branch microchannel 90 connects to second connection tube 50.

A single-cell trap 110 is disposed in base microchannel 80, distal to convergence point 95. As will hereinafter be discussed, single-cell trap 110 allows a single cell to be captured, and thereby isolated, from a group of cells, and thereafter selectively released, for transfer to a desired location .

More particularly, and looking now at Figs. 5-8, single-cell trap 110 is disposed as "an island" in base microchannel 80, whereby to divide the base microchannel into a wide path 115 and a narrow path 120. Wide path 115 is sized so as to be able to pass cells and fluid therethrough. Narrow path 120 is sized so as to pass only fluid therethrough. Single- cell trap 110 comprises a body 125 comprising a flow diverter 130 and a well 135. Flow diverter 130 is spaced from the side wall of base microchannel 80 and diverts the flow passing by single-cell trap 110 into either wide path 115 or narrow path 120. Well 135 is disposed on the distal side of body 125, outboard of flow diverter 130 (and proximal to the distalmost portion of flow diverter 130) and, together with flow diverter 130 and the side wall of base microchannel 80, provides a seat for one individual cell. Note that the entrance of well 135 (Fig. 6) is larger than narrow path 120 which exits well 135, with well 135 being sized to receive a cell and with narrow path 120 being sized too small to receive a cell, so that a cell may enter well 135 but not exit well 135 via narrow path 120. In other words, single-cell trap 110 is configured so that the single-cell trap has a wider opening on the side of the single-cell trap which faces the source of the cell slurry and a smaller opening on the side of the single-cell trap which faces away from the source of the cell slurry, with the wider opening being sized to receive a cell and with the smaller opening being too small to receive a cell . In one preferred form of the invention, single- cell pipette tip 15 may have a length of 5 mm and a distal tip width of 300 pm. Base microchannel 80 may have a width of 40 pm, wide path 115 may have a width of 22 pm, narrow path 120 may have a width of 3 pm, flow diverter 130 may have a width of 6 pm and well 135 may have a width of 12 pm and a depth of 10 pm.

Single-cell pipette tip 15 is preferably provided by first designing the single-cell pipette tip with CAD software and then fabricating the single-cell pipette tip using photolithography and

polydimethylsiloxane (PDMS) molding techniques. Among other things, and as will hereinafter be discussed, PDMS is a preferred material due to (i) the poor adhesion of cells thereto, (ii) its excellent

elasticity, and (iii) its good optical transparency.

On account of the foregoing construction, and looking now at Figs. 9A-9D, when a slurry of cells C passes proximally through base microchannel 80 (i.e., from distal tip 75 toward convergence point 95), flow diverter 130 causes some of the flow to pass down wide path 115 and some of the flow to pass down narrow path 120. Significantly, as the cells C in the slurry encounter flow diverter 130 of single-cell trap 110, most of the cells C follow wide path 115 and pass by single-cell trap 110. See Figs. 9A and 9B. However, in some instances, a single cell C will be diverted into well 135 of single-cell trap 110. See Fig. 9C. Note that cells C may cluster in wide path 115

adjacent to flow diverter 130 as they try to move through wide path 115, and this clustering of cells may assist in a single cell being diverted into well 135. Thereafter, and looking now at Fig. 9D, the captured cell C may be selectively released from single-cell trap 110 (e.g., for transfer to a desired location) by flowing fluid distally through base microchannel 80 (i.e., by flowing fluid from

convergence point 95 to distal tip 75) .

Note that there are two potential flow paths around single-cell trap 110: the capture path (i.e., narrow path 120) and the bypass path (i.e., wide path 115) . Flow profile simulations show that the flow rate along the bypass path (i.e., wide path 115) is much larger than the flow rate along the capture path (i.e., narrow path 120), indicating that single cells prefer to flow along the bypass path rather than along the capture path, thereby generally resulting in a failed capture. It has been found that single cell capture can only occur occasionally when the cell concentration is lower than about 10⁵ cells/mL.

However, when the cell concentration is higher than about 10⁶ cells/mL, single cell capture can occur much more often as the bypass path (i.e., wide path 115) is frequently occupied by other cells at this

concentration level. In other words, at this higher concentration level, it is more likely that the cells may cluster in the bypass path (i.e., wide path 115) as the cells try to move through the bypass path

(i.e., wide path 115), and this clustering of cells may assist in a single cell being diverted into well 135 of single-cell trap 110.

A variety of factors have been identified which can affect single cell capture efficiency, including fluid resistance ratios, aspiration times, and cell concentrations. Experimental results reveal that the highest capture efficiency is achieved when the width of the bypass path (i.e., wide path 115) is

approximately 1.67 times larger than the diameter of the cell to be captured. A decrease in the ratio of fluid resistance along the capture path (R_c) to the fluid resistance along the bypass path (i¾) can also increase the probability of single cell capture by the single-cell trap 110. In other words, and looking now at Fig. 10, by increasing the length of wide path 115 relative to narrow path 120, whereby to increase the fluid resistance R_b of wide path 115 relative to the fluid resistance R_c of narrow path 120, the probability of single cell capture can be increased.

It has also been found that longer aspiration times are beneficial to improving single cell capture efficiency.

Single cell capture efficiency can reach up to 96.7% where the ratio of the fluid resistance along the capture path to the fluid resistance along the bypass path (R_c/R_b) is 36, the aspiration time is 5 seconds, and the cell suspension has a concentration of 10⁷/mL.

By way of further example but not limitation, Fig. 10A shows a simple model of single-cell capture by the single-cell trap 110; Fig. 10B shows single- cell capture efficiency under various fluid-resistance rations R_c/R_b; Fig. IOC shows single-cell capture efficiency under various aspiration times and cell concentrations; Fig. 10D shows single-cell capture efficiency with various bypass path widths; and Fig. 10E shows single-cell capture efficiency with various bypass path lengths.

Use Of The Novel Single-Cell Pipette Assembly To Isolate An Individual Cell From A Group Of Cells And Transfer That Isolated Cell To A Desired Location Figs. 11-19 show how novel single-cell pipette assembly 5 may be used to isolate an individual cell from a group of cells and transfer that isolated cell to a desired location.

More particularly, in one preferred method of use, first connection tube 40 and second connection tube 50 are first mounted to body 20 of single-cell pipette handle 10, if they are not already mounted to body 20, so that first connection tube 40 is in fluid communication with first pressure channel 25 and second connection tube 50 is in fluid communication with second pressure channel 30. See Figs. 11 and 12. It will be appreciated that when first connection tube 40 is in fluid communication with first pressure channel 25 and second connection tube 50 is in fluid communication with second pressure channel 30, first connection tube 40 effectively constitutes an

extension of first pressure channel 25 and second connection tube 50 effectively constitutes an

extension of second pressure channel 30.

Then first connection tube 40 and second

connection tube 50 are primed with primer solution, i.e., by positioning the distal ends of first

connection tube 40 and second connection tube 50 in primer solution 140, and then retracting first plunger 55 within first passageway 35 and second plunger 60 within second passageway 45. See Fig. 13. Note that the primer fluid is not pulled into either first passageway 35 or second passageway 45 of body 20 of single-cell pipette handle 10. As a result, first passageway 35 and second passageway 45 of body 20 of single-cell pipette handle 10 are uncontaminated .

Then the distal ends of first connection tube 40 and second connection tube 50 are withdrawn from primer solution 140. See Fig. 14.

Next, single-cell pipette tip 15 is mounted to single-cell pipette handle 10 by connecting first connection tube 40 to first coupling 100 of single- cell pipette tip 15 and by connecting second connection tube 50 to second coupling 105 of single- cell pipette tip 15. See Fig. 15. In this respect it will be appreciated that the excellent elasticity of PDMS facilitates the creation of a good seal at the interface between first connection tube 40 and first coupling 100 of single-cell pipette tip 15 and at the interface between second connection tube 50 and second coupling 105 of single-cell pipette tip 15, whereby to minimize the leakage of gases and liquids. Then, Y- shaped microchannel 70 in single-cell pipette tip 15 is primed using the primer solution in first

connection tube 40 and second connection tube 50, i.e., by advancing first plunger 55 within first passageway 35 and second plunger 60 within second passageway 45. See Fig. 16. Preferably first plunger

55 is advanced sufficiently within first passageway 35 so as to eject at least some primer solution out of the distal end of single-cell pipette tip 15, and second plunger 60 is advanced sufficiently within second passageway 45 so as to eject at least some primer solution out of the distal end of single-cell pipette tip 15, whereby to ensure that Y-shaped microchannel 70 is completely filled with primer solution .

Next, distal tip 75 of single-cell pipette 15 is disposed in a slurry of cells 145, and negative pressure is applied to first pressure channel 25, i.e., by withdrawing first plunger 55 within first passageway 35, whereby to draw the slurry of cells proximally up into base microchannel 80 and into first branch microchannel 85. See Fig. 17. This action causes a single cell from the slurry to be captured in single-cell trap 110, e.g., in the manner shown in

Figs. 9A-9C. Note that neither primer fluid 140 nor slurry of cells 145 is/are pulled into either first passageway 35 or second passageway 45 of body 20 of single-cell pipette handle 10. As a result, first passageway 35 and second passageway 45 of body 20 of single-cell pipette handle 10 remain uncontaminated . Then distal tip 75 of single-cell pipette 15 is withdrawn from the slurry of cells 145 and is

positioned in a wash solution 150. Negative pressure is applied to first pressure channel 25, i.e., by withdrawing first plunger 55 within first passageway 45, so that the wash solution flushes the slurry of cells out of base microchannel 80, with the captured cell remaining in single-cell trap 110. See Fig. 18. Again, note that neither primer fluid 140 nor slurry of cells 145 nor wash solution 150 is/are pulled into either first passageway 35 or second passageway 45 of body 20 of single-cell pipette handle 10. As a result, first passageway 35 and second passageway 45 of body 20 of single-cell pipette handle 10 remain uncontaminated .

If desired, the successful capture of a cell in single-cell trap 110 can be confirmed visually. By way of example but not limitation, single-cell pipette tip 15 can be placed beneath a microscope and single- cell trap 110 visually examined to confirm that a cell has been captured in single-cell trap 110. In this respect it will be appreciated that the good optical transparency of PDMS facilitates visual examination of single-cell trap 110, and of a cell captured in single-cell trap 110, by microscopic examination.

In some cases it may be desirable to dismount single-cell pipette tip 15 from single-cell pipette handle 10 in order to facilitate viewing under a microscope. In this case, first connection tube 40 of single-cell pipette handle 10 is disconnected from first coupling 100 of single-cell pipette tip 15 and second connection tube 50 of single-cell pipette handle 10 is disconnected from second coupling 105 of single-cell pipette tip 15, and then single-cell pipette tip 15 is positioned beneath the microscope. In one preferred form of the invention, and looking now at Figs. 19A and 19B, single-cell pipette tip 15 may be formed with a flat surface 152 for enabling single-cell pipette tip 15 to remain stationary while being viewed using a microscope.

Thereafter, when the single cell captured in single-cell trap 110 is to be transferred to a desired location, positive pressure is applied to second pressure channel 30, i.e., by advancing second plunger 60 distally through second passageway 45, so that the primer solution in second connection tube 50 is flushed through second branch microchannel 90, into base microchannel 80 and then out distal tip 75 of single-cell pipette tip 15, whereby to flush the captured cell out of single-cell trap 110 (e.g., in the manner shown in Fig. 9D) and hence out distal tip 75 of single-cell pipette tip 15 and into (or onto) an appropriate receptacle (or support) 155 (e.g., a common 96- or 384-well plate, a cell culture dish, a vial, a microscope slide, etc.) .

The entire process can be completed within 10 seconds .

Thus it will be seen that single-cell pipette assembly 5 may be used to obtain single cells directly from a cell suspension. Single-cell pipette assembly

5 is characterized by operational simplicity, high efficiency and low cost.

Significantly, single-cell pipette assembly 5 does not harm the individual cell captured by single- cell trap 110, so that single-cell pipette assembly 5 can be used for live single cell isolation and

transfer .

Also significantly, with the present invention, single cell isolation and transfer is effected using an operation which is generally similar to the

operation used to transfer liquid with an Air

Displacement Pipette (ADP) , thereby greatly minimizing operational training and cost. Using The Novel Single-Cell Pipette Assembly To

Isolate And Transfer Additional Individual Cells By

Repeating The Foregoing Process Single-cell pipette assembly 5 may be used to isolate and transfer additional individual cells by repeating the foregoing process. In this respect it should be appreciated that, where it is desirable to avoid cross-contamination between cell samples, first connection tube 40, second connection tube 50 and single-cell pipette tip 15 may be dismounted from body 20 of single-cell pipette handle 10 and replaced.

Note that inasmuch as primer solution 140, slurry of cells 145 and wash solution 150 is/are never drawn up into first passageway 35 or second passageway 45 of body 20 of single-cell pipette handle 10, first passageway 35 and second passageway 45 of body 20 of single-cell pipette handle 10 remain uncontaminated . Thus, replacing first connection tube 40, second connection tube 50 and single-cell pipette tip 15 between cell samples prevents cross-contamination even though the same single-cell pipette handle 10 may be reused .

Additional Constructions

1. Alternative Means For Applying Positive Or

Negative Pressure To First Pressure Channel 25 And Second Pressure Channel 30

The single-cell pipette assembly 5 shown in Figs. 1-19 comprises first plunger 55 and second plunger 60 for applying positive or negative pressure to first pressure channel 25 and second pressure channel 30, respectively. However, if desired, other means may be provided for applying positive or negative pressure to first pressure channel 25 and second pressure channel 30, respectively. By way of example but not

limitation, and looking now at Figs. 20-28, a first screw drive 55 and a second screw drive 60 may be provided for applying positive or negative pressure to first pressure channel 25 and second pressure channel 30, respectively.

2. Alternative Pressure-Applying/Vacuum- Applying Apparatus

If desired, single-cell pipette handle 10 may be replaced by alternative pressure-applying/vacuum- applying apparatus comprising a first pressure channel capable of applying positive and negative pressure to first coupling 100 of single-cell pipette tip 15, and a second pressure channel capable of applying positive and negative pressure to second coupling 105 of cell pipette tip 15, e.g., an appropriate multi-channel pressure/vacuum source.

3. Configuring Single-Cell Pipette Assembly 5 To Isolate And Transfer More Than One Cell With Each Cycle Of The Single-Cell Pipette Assembly

In another form of the invention, single-cell pipette assembly 5 can be configured to isolate and transfer more than one cell with each cycle of the single-cell pipette assembly. More particularly, where it is desired to transfer N cells with each cycle of single-cell pipette assembly 5, N single-cell traps 110 are positioned in base microchannel 80 of single-cell pipette tip 15. As a result, with each cycle of single-cell pipette assembly 5, N cells are captured and transferred. See Fig. 28A. 4. Mounting Multiple Single-Cell Pipette Tips

To Single-Cell Pipette Handle 10

Furthermore, if desired, single-cell pipette assembly 5 may be configured to mount multiple single- cell pipette tips 15 to single-cell pipette handle 10, so that single-cell pipette assembly 5 may

simultaneously capture, and then simultaneously transfer, a plurality of cells, with those cells being spatially separated from one another. In this

situation, single-cell pipette handle 10 is configured to receive multiple pairs of first connection tube 40, second connection tube 50 and, for each pair of first connection tube 40, second connection tube 50, to connect first connection tube 40 to first pressure channel 25 and second connection tube 50 to second pressure channel 30, respectively. See Figs. 29-32.

In Figs. 29 and 30, single-cell pipette assembly 5 comprises four single-cell pipette tips 15 mounted to a single-cell pipette handle 10, with the single- cell pipette tips 15 being arranged in a linear configuration (when seen in end view); and in Figs. 31 and 32, single-cell pipette assembly 5 comprises eight single-cell pipette tips 15 mounted to a single-cell pipette handle 10, with the single-cell pipette tips

15 being arranged in a linear configuration (when seen in end view) .

It will be appreciated that more or less single- cell pipette tips 15 may be mounted to a single-cell pipette handle 10.

It will also be appreciated that the plurality of single-cell pipette tips 15 mounted to a single-cell pipette handle 10 may be arranged in configurations other than linear (when seen in end view) . By way of example but not limitation, the plurality of single- cell pipette tips 15 (mounted to a single-cell pipette handle 10) may be arranged in a two dimensional matrix configuration (when seen in end view), e.g., an 8 x 12 matrix configuration, or a 16 x 24 matric

configuration, etc. By way of further example but not limitation, the plurality of single-cell pipette tips 15 may be arranged in a circular configuration (when seen in end view) . If desired, the plurality of single-cell pipette tips 15 may be secured to one another, or formed integral with one another, so as to form a singular construction .

5. Forming A Single-Cell Pipette Tip Which Is The Equivalent Of Multiple Single-Cell Pipette Tips Mounted Together As A Singular Construction But Modified So As To Share A Single Distal End Inlet/Outlet

In still another preferred form of the present invention, and looking now at Fig. 33, there is provided a single-cell pipette tip 15 comprising a body 65 having a plurality of Y-shaped microchannels 70 formed therein, wherein the plurality of Y-shaped microchannels 70 share a common distal end

inlet /outlet . More particularly, in this form of the invention, body 65 has a center opening 160 leading to a distal tip (not shown in Fig. 33 but analogous to the distal tip 75 shown in Figs. 4 and 5) . As discussed above, each Y-shaped microchannel 70 comprises a base microchannel 80, a first branch microchannel 85 and a second branch microchannel 90, with base microchannel 80, first branch microchannel 85 and second branch microchannel 90 converging at a convergence point 95. Base microchannel 80 extends from convergence point 95 to center opening 160 and then to distal tip 75. First branch microchannel 85 extends from convergence point 95 to a first coupling 100, which is configured for connection to first pressure channel 25 of a single-cell pipette handle 10 (or to another first pressure channel providing a positive/negative pressure source) . Second branch microchannel 90 extends from convergence point 95 to a second coupling 105, which is configured for

connection to second pressure channel 30 of a single- cell pipette handle 10 (or to another second pressure channel providing a positive/negative pressure

source) . Again, a single-cell trap 110 (shown

schematically in Fig. 33 for reasons of scale) is disposed in each base microchannel 80, distal to convergence point 95. As discussed above, each single-cell trap 110, working in conjunction with Y- shaped microchannel 70 and appropriate sources of positive/negative pressure operating in an appropriate manner (including sequence), allows a single cell to be captured, and thereby isolated, from a group of cells, and thereafter selectively released, for transfer to a desired location.

Thus it will be seen that the single-cell pipette tip 15 shown in Fig. 33 provides the equivalent of multiple single-cell pipette tips 15 mounted together as a singular construction but modified so as to share a common distal end inlet /outlet .

The single-cell pipette tip 15 shown in Fig. 33 can be highly advantageous since a single distal tip (not shown in Fig. 33 but analogous to distal tip 75 shown in Figs. 4 and 5) and a single center opening 160 can be used as the distal end inlet/outlet for each of the Y-shaped microchannels 70 provided in single-cell pipette tip 15. Thus, if desired, the aforementioned priming step (with primer solution 140), single-cell capture step (with slurry of cells 145 and single-cell traps 110) and wash step (with wash solution 150) can be simultaneously effected for each of the Y-shaped microchannels 70 in body 65 of single-cell pipette tip 15 using a common source of primer solution 140, a common source of a slurry of cells 145 and/or a common source of a wash solution 150; and then the aforementioned cell-release step (by flushing with primer solution 140) can be individually effected for each of the Y-shaped microchannels 70 (i.e., by selectively applying positive pressure to each of the second couplings 105, one at a time) . 6. Culturing A Cell After The Cell Has Been

Captured In A Single-Cell Trap And Before The Cell Is Released From A Single-Cell Trap It is also possible to culture a cell after the cell has been captured in a single-cell trap 110 and before the cell is released from single-cell trap 110.

By way of example but not limitation, with the single- cell pipette tip shown in Fig. 33, after an individual cell has been seated in a single-cell trap 110 of an individual Y-shaped microchannel 70, the cell may be cultured (e.g., for one to three hours) and thereafter released from the single-cell trap 110 of that Y- shaped microchannel. See Fig. 34. In this form of the invention, primer solution 140 may preferably comprise trypsin, since trypsin can facilitate the release of a cultured cell from single-cell trap 110.

Using A "Single-Channel" Air Displacement Pipette (ADP) , Together With A Dual-Channel Adapter, In Place

Of The "Dual-Channel" Air Displacement Pipette (ADP) In the preceding description, single-cell pipette handle 10 comprises a "dual-channel" Air Displacement Pipette (ADP) device, i.e., single-cell pipette handle 10 comprises (i) a first pressure channel 25, a first passageway 35 and a first plunger 55 for applying negative pressure (-P) or positive pressure (+P) to a first connection tube 40, and (ii) a second pressure channel 30, a second passageway 45 and a second plunger 60 for applying negative pressure (-P) or positive pressure (+P) to a second connection tube 50.

However, if desired, a "single-channel" Air

Displacement Pipette (ADP) , together with a dual- channel adapter, may be used in place of the "dual- channel" Air Displacement Pipette (ADP) .

More particularly, and looking now at Figs. 35- 37, there is shown a single-cell pipette handle 210 which comprises a body 220 and a pressure channel 225. In one preferred form of the invention, pressure channel 225 comprises a passageway 235 extending through body 220 and terminating in an end opening 227. A plunger 255 is movably disposed in passageway 235, such that movement of plunger 255 in passageway 235 can apply positive or negative pressure to opening 227. It should be appreciated that, if desired, single-cell pipette handle 210 may comprise a

traditional "single-channel" Air Displacement Pipette (ADP) of the sort well known in the art for providing a negative pressure produced by moving plunger 255 in a first (i.e., proximal) direction, and a positive pressure produced by moving plunger 255 in a second, opposite (i.e., distal) direction.

Still looking now at Figs. 35-37, there is shown a dual-channel adapter 237 which generally comprises a body 239 which defines a first passageway 240

terminating in an opening 241 and a second passageway 250 terminating in an opening 251.

In use, dual-channel adapter 237 is mounted to single-cell pipette tip 15 so that first passageway 240 of dual-channel adapter 237 is connected to first coupling 100 of single-cell pipette tip 15 and second passageway 250 of dual-channel adapter 237 is

connected to second coupling 105 of single-cell pipette tip 15. Single-cell isolation can be achieved in 3 steps using single-cell pipette handle 210, dual-channel adapter 237 and single-cell pipette tip 15.

Step 1: Single-cell Capture And Washing. After applying negative pressure (-P) to first passageway

240 of dual-channel adapter 237 using single-cell pipette handle 210 (e.g., by moving plunger 255 proximally in passageway 235), the distal end of single-cell pipette tip 15 is rapidly immersed into a cell suspension (e.g., for 10-20 seconds) to allow cells to be aspirated into base microchannel 80 of single-cell pipette tip 15. During this process, one cell may be randomly captured by single-cell trap 110 in single-cell pipette tip 15. According to the fluid distribution in the Y-shaped microchannel 70, cells only flow into first branch microchannel 85 (e.g., in a manner similar to that shown in Fig. 9B) and no cells will flow into second branch microchannel 90. The residual cells in base microchannel 80 are washed out by dipping single-cell pipette tip 15 into cell-free liquids while moving up and down several times. Meanwhile, under the effect of the negative pressure (-P) produced by single-cell pipette handle 210, all of the uncaptured cells within base

microchannel 80 will be rapidly aspirated into first branch microchannel 85 and first passageway 240 of dual-channel adapter 237. In order to obtain optimum pressure control for single-cell capture, three types of negative pressure (-P) were investigated, including -100 i^~L, -150 i^~L, and -200 iL (which are consistent with commercially-available single-cell pipette handles which typically have a volume range of 20-200iL) . The liquid velocity in base microchannel 80 was measured. Results showed that 200 iL was generally preferable for cell capture.

Step 2: Single-Cell Identification. Due to easy disassembly between single-cell pipette handle 210 and dual-channel adapter 237, the combined dual-channel adapter 237 and single-cell pipette tip 15 are

separated from single-cell pipette handle 210 and put on a Petri dish for microscopic identification. See Fig. 38. With the good optical transparency afforded by PDMS and the flat bottom 152 of single-cell pipette tip 15, the single cell captured by single-cell trap 110 can be easily and clearly identified under a common inverted microscope.

Step 3: Single-Cell Release And Transfer. Due to the poor adhesion of cells on PDMS, the single cell captured by single-cell trap 110 can be easily

released from single-cell trap 110 by applying

positive pressure (+P) to second branch microchannel 90 (e.g., in a manner similar to that shown in Fig. 9D) . This is done by connecting outlet 227 of single- cell pipette handle 210 to second passageway 250 of dual-channel adapter 237 (which is attached to second coupling 105 of single-cell pipette tip 15) and by moving plunger 255 distally in passageway 235. In order to allow the single cell captured in single-cell trap 110 to be flowed out within the shortest time, a positive pressure (+P) of 200 iL is preferably applied to second passageway 250 of dual-channel adapter 237, e.g., for 5-10 seconds. This causes positive pressure (+P) to be applied to second branch microchannel 90 of single-cell pipette tip 15 so as to release the identified single cell from single-cell pipette tip 15 and cause it to be transferred to another container.

Of course, it will be appreciated by those skilled in the art that single-cell pipette handle 210, dual-channel adapter 237 and single-cell pipette tip 15 may also be used in other ways to isolate an individual cell from a group of cells and transfer that isolated cell to a desired location.

Alternative Form Of Single-Cell Trap The success rate for capturing a cell in the aforementioned single-cell trap 110 can vary according to the concentration of cells in a cell slurry. By way of example but not limitation, when the cell concentration is higher than about 10⁶ cells/mL, single cell capture tends to occur much more often since the bypass path (i.e., wide path 115, see Figs. 9A-9D) is frequently occupied by other cells at this

concentration level. However, when the cell concentration is lower than about 10 cells/mL, single cell capture typically occurs less often.

Accordingly, in another form of the present invention, and looking now at Figs. 39-41, the

aforementioned single-cell trap 110 is replaced by a single-cell trap 310.

More particularly, in this form of the invention, base microchannel 80 has a serpentine configuration wherein two serial lengths 80A, 80B of base

microchannel 80 extend adjacent to one another, and single-cell trap 310 is formed in the side wall 80C separating length 80A from length 80B. More

particularly, in this form of the invention, single- cell trap 310 comprises a passageway comprising an inlet 315 (e.g., a recess or well) communicating with length 80A and an outlet 320 (e.g., an opening) communicating between inlet 315 and length 80B. Note that inlet 315 (which essentially acts as the recess or well for seating a cell) is larger than outlet 320 (which essentially acts as an outlet from the recess or well which seats a cell), with inlet 315 and outlet 320 being appropriately sized so that a cell may enter inlet 315 but not exit via outlet 320. In other words, single-cell trap 310 is configured so that it has a wider inlet (i.e., opening) on the side of the single-cell trap which faces the source of the cell slurry and a smaller outlet (i.e., opening) on the side of the single-cell trap which faces away from the source of the cell slurry, with the wider inlet (i.e., opening) being sized to receive a cell and with the smaller outlet (i.e., opening) too small to receive a cell. Note also that inlet 315 is sized to be

compatible with a range of different cells (e.g., NIH

3T3 cells having a diameter of 13-18 pm, K562

lymphoblasts having a diameter of 12-17 pm, NK-92 cells having a diameter of 11-20 pm) and outlet 320 is sized to be small enough to ensure that a single cell seated within inlet 315 is unable to pass out outlet

320 (e.g., outlet 320 may have a diameter of 3 pm) . It has been found that the configuration of single- cell trap 310 yields a higher capture rate than the configuration of single-cell trap 110. This is believed to be due to the fact that the fluid

resistance in length 80A of base microchannel 80 is a little higher than that in single-cell trap 310, so that a single cell preferentially flows into the single-cell trap. By way of example but not

limitation, it has been found that a cell capture rate of 96.7% can be achieved with cell concentrations of 10 cells per pL, and a cell capture rate of 76.7% can be achieved with cell concentrations of 10 cells per pL.

Fig. 42 shows exemplary dimensions for a single- cell trap 310 formed in a side wall 80C separating a length 80A of base microchannel 80 from a length 80B of base microchannel 80. Incorporation By Reference

Still further details regarding novel aspects of the present invention are set forth in Kai Zhang et al . , "Single-Cell Isolation by Modular Single-Cell Pipette for RNA-Sequencing", Lab On A Chip, Royal Society Of Chemistry, November 2, 2016, which

publication is hereby incorporated herein by

reference .

Modifications Of The Preferred Embodiments

It should be understood that many additional changes in the details, materials, steps and

arrangements of parts, which have been herein

described and illustrated in order to explain the nature of the present invention, may be made by those skilled in the art while still remaining within the principles and scope of the invention.

Claims

What Is Claimed Is:

1. Apparatus for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, said apparatus comprising: a single-cell pipette tip, said single-cell pipette tip comprising:

a structure having a distal end;

microchannel .

2. Apparatus according to claim 1 wherein said base microchannel has a serpentine configuration wherein first and second serial lengths of said base microchannel extend adjacent to one another, separated by a side wall .

3. Apparatus according to claim 2 wherein said single-cell trap comprises a passageway extending through said side wall.

4. Apparatus according to claim 3 wherein said single-cell trap comprises a passageway comprising an inlet and an outlet, wherein said inlet communicates with said first serial length of said base

microchannel and said outlet communicates with said second serial length of said base microchannel.

5. Apparatus according to claim 4 wherein said inlet has a larger width than said outlet.

6. Apparatus according to claim 5 wherein said inlet has a width large enough to receive a cell therein and said outlet has a width too small to pass a cell therethrough.

7. Apparatus according to claim 6 wherein said inlet has a length of approximately 18 pm and a width of approximately 17 pm, and further wherein said outlet has a length of approximately 8 pm and a width of approximately 3 pm.

8. Apparatus according to claim 1 further comprising :

a single-cell pipette handle comprising a

pressure channel for providing negative pressure or positive pressure; and

a dual-channel adapter comprising a first channel and a second channel, said first channel of said dual- channel adapter being connectable to said pressure channel of said single-cell pipette handle and to said first branch microchannel of said single-cell pipette tip, and said second channel of said dual-channel adapter being connectable to said pressure channel of said single-cell pipette handle and to said second branch microchannel of said single-cell pipette tip.

9. Apparatus according to claim 8 wherein said dual-channel adapter is separable from said single- cell pipette handle.

10. Apparatus according to claim 9 wherein said dual-channel adapter is separable from said single- cell pipette tip.

11. Apparatus according to claim 1 wherein said single-cell pipette tip comprises a transparent material .

12. Apparatus according to claim 11 wherein said transparent material comprises polydimethylsiloxane (PDMS) .

13. Apparatus according to claim 1 wherein said single-cell pipette tip comprises a flat surface for enabling said single-cell pipette tip to remain stationary .

14. A method for isolating an individual cell from a group of cells and transferring that isolated cell to a desired location, said method comprising: providing a single-cell pipette tip, said single- cell pipette tip comprising:

a structure having a distal end;

microchannel ;

positioning said base microchannel in a slurry of cells ;

positioning said base microchannel in a wash solution;

15. A method according to claim 14 wherein said base microchannel has a serpentine configuration wherein first and second serial lengths of said base microchannel extend adjacent to one another, separated by a side wall .

16. A method according to claim 15 wherein said single-cell trap comprises a passageway extending through said side wall.

17. A method according to claim 16 wherein said single-cell trap comprises a passageway comprising an inlet and an outlet, wherein said inlet communicates with said first serial length of said base

18. A method according to claim 17 wherein said inlet has a larger width than said outlet.

19. A method according to claim 18 wherein said inlet has a width large enough to receive a cell therein and said outlet has a width too small to pass a cell therethrough.

20. A method according to claim 19 wherein said inlet has a length of approximately 18 pm and a width of approximately 17 pm, and further wherein said outlet has a length of approximately 8 pm and a width of approximately 3 pm.

21. A method according to claim 14 further comprising :

a single-cell pipette handle comprising a

pressure channel for providing negative pressure or positive pressure; and

22. A method according to claim 21 wherein said dual-channel adapter is separable from said single- cell pipette handle.

23. A method according to claim 22 wherein said dual-channel adapter is separable from said single- cell pipette tip.

24. A method according to claim 14 wherein said single-cell pipette tip comprises a transparent material .

25. A method according to claim 24 wherein said transparent material comprises polydimethylsiloxane (PDMS) .

26. A method according to claim 14 wherein said single-cell pipette tip comprises a flat surface for enabling said single-cell pipette tip to remain stationar .

27. A method according to claim 14 further comprising :

visually examining said single-cell trap to determine if a single cell has been captured in said single-cell trap.

28. A method according to claim 27 wherein a microscope is used to visually examine said single- cell trap.

RECTIFIED (RULE 91) - ISA/US