WO2003050231A1

WO2003050231A1 - Pipette apparatus

Info

Publication number: WO2003050231A1
Application number: PCT/US2002/039996
Authority: WO
Inventors: Pi-Yao Aileen Liu
Original assignee: Pi-Yao Aileen Liu
Priority date: 2001-12-13
Filing date: 2002-12-13
Publication date: 2003-06-19
Also published as: AU2002360603A1

Abstract

Apparatus and method for delivering and withdrawing material from a container includes a first conduit (1004) with tip (1006) and a second conduit (1010) with tip (1008). Vacuum ports (1020, 1023) are coupled to each conduit and a medium supply port (1101) is coupled to the first conduit (1004). The first tip (1006) is mounted to provide movement relative to the second tip (1008) to prevent cross contamination between the fluid delivered and the fluid withdrawn.

Description

Pipette Apparatus

Field of the Invention

The present invention relates to the field of biological cellular cultures. More particularly, it relates to a novel apparatus and method for changing a liquid culture medium used to cultivate cell cultures with replacement medium, wherein methods, devices and systems for removing and delivering liquid media utilize separate conduits, one conduit which is moveable in order to avoid cross contamination between old and new media.

Background of the Invention

Changing culture medium is a routine and labor-intensive process of isolating cell culture, typically performed on a daily basis. Traditionally, the procedures of changing culture medium include at least two separate steps: 1) Aspiration or removal of the old medium from a culture vessel by means of vacuum suction or pipetting; a process of dipping an open-ended tube into the liquid medium, and withdrawing the tube and liquid within the tube from the medium for disposal.

2) Addition of replacement medium to the culture vessels using another tool, usually a pipette, to transfer medium from a liquid container to the culture vessels. Generally, the process for the conventional operation of changing medium is described below. If employing a vacuum means: aspirate the old medium, change the tool, draw the replacement medium, and release the replacement medium to the culture vessels. If pipetting: draw the old medium, dispose of it, draw replacement medium, and release the replacement medium to the culture vessels. The operator may or may not change pipettes to isolate the replacement medium from potential intermixing of residue from the old medium contained within the pipette.

These procedures involve many actions, multiple pauses, and frequent opening of culture vessels and medium containers. Furthermore the potential exists for the prolonged exposure of cultured cells to air, particularly when a large volume of medium or a large number of vessels need to be changed. Therefore, the process is time consuming in the least and potentially harmful to the cultured cells due to their prolonged exposure to air as well as significantly increasing the risk of contamination due to the repetitive actions necessary.

One proposed solution is shown in U.S. 5,874,296. The patent describes a liquid medium exchange and dispensing product used to exchange old culture media with new media. The structure shown in the '296 patent may be subject to cross contamination because the same conduit is used for new and old medium.

Thus, it is desirable to provide an improved pipette that avoids cross contamination by providing methods, devices and systems for removing and delivering liquid media utilizing separate conduits, one of which is moveable.

Other examples of pipette devices are set forth in the following U.S. patents:

U.S. Patent No. 4,015,942 U.S. Patent No. 4,635,665 U.S. Patent No. 5,120,305

U.S. Patent No. 5,542,918 U.S. Patent No. 5,807,524 U.S. Patent No. 5,851,491

Of these, U.S. Patent No. 4,015,942 discloses an apparatus for delivering liquid to a plurality of containers and removing contents therefrom, comprising a plurality of pairs of tubes each tube having or being connected to a constrictible tube portion. A first tube of each pair is adapted to deliver liquid to a respective container from a liquid supply and the second tube of each pair is adapted for connection between suction means and a tube portion adapted to project into a respective container to remove liquid therefrom. Tube constricting means are provided and arranged to constrict said tube portions alternately whereby respectively liquid is delivered to each container and contents removed therefrom.

U.S. Patent No. 4,635,665 discloses an apparatus that comprises holding a pipe assembly by means of a holding means. The pipe assembly is consisted of a pouring pipe which acts to pour a wash liquid in micro wells of a container, and a suction pipe which acts to suck said waste wash liquid from said micro wells. The pipe assembly is formed of a dual-pipe consisting of a suction pipe and a pouring pipe disposed inside the suction pipe. The forward end opening portions of both pipes are located on substantially the same plane. U.S. Patent No. 5,120,305 discloses a method and apparatus for delivering and/or withdrawing fluids. For example, blood may be salvaged from a body cavity of a patient, in which a wand is provided having separate conduits for fluids. The apparatus is adapted for surgical use, and most particularly when a partial vacuum is drawn through the conduits, such that operation of the valve can exert a partial vacuum on one or more of the conduits. The entry ends of the conduits are disposed adjacent each other, with the conduits being joined as a wand.

U.S. Patent No. 5,807,524 discloses a replaceable pipette tip comprising a pipette surface contamination protector comprising an upwardly extending projection connected by the shoulder to the proximal end portion for laterally spacing the pipette device from a tube which contains or which is to receive the sample fluid to prevent contamination of an outer surface of the pipette device by contact with the tube into which the pipette tip is inserted.

U.S. Patent No. 5,851,491 discloses a filter for a pipette tip comprising a plurality of vertically oriented cylindrical micro fibers cohesively bundled in adjoining columns. The compression of the fibers creates vertically oriented pores mterstitially between the micro fibers, each pore having a pore size at various points within the filter. Each filter has an equal predetermined density of micro fibers per square millimeter in its uncompressed state, such that when the filter is compressed, its pore sizes will be consistent with another filter used in a pipette tip of the same size and shape. However, none support a pipette apparatus that eliminates cross-contamination.

Accordingly, there exists a need to provide an improved pipette that avoids cross contamination by providing methods, devices and systems for removing and delivering liquid media utilizing separate conduits, one of which is moveable. Summary of the Invention

The present invention provides an improved pipette apparatus that eliminates cross contamination, by incorporating a moveable external conduit element that is used to convey away old medium. The apparatus, which can be used in the delivery of medium to and the withdrawal of material from containers, includes a vacuum source, a replacement medium source, and first and second conduits, wherein the conduits further include a first conduit with a tip having an orifice, for delivery of liquid to a container, and a second conduit external to the first conduit, including a tip with an orifice, for withdrawal of material from a container by suction. The apparatus also includes support elements for mounting the conduits, wherein the tip of the second conduit may be moved relative to the tip of the first conduit to prevent cross-contamination between old and new media.

Brief Description of the Drawings

FIGS. 1-5 are schematic diagrams illustrating a pipette typical of the prior art. FIG. 6 shows an apparatus constructed in accordance with the present invention.

FIG. 7 shows an embodiment of the apparatus constructed in accordance with the present invention.

FIG. 8a shows an embodiment of the apparatus constructed in accordance with the present invention. FIG. 8b shows a portion of the apparatus constructed in accordance with the present invention.

FIG. 9 shows an embodiment of the apparatus constructed in accordance with the present invention.

FIG. 10 shows an embodiment of the apparatus constructed in accordance with the present invention. FIG. 11 shows an embodiment of the head assembly constructed in accordance with the present invention.

FIG. 10 shows an embodiment of the apparatus constructed in accordance with the present invention.

Detailed Description of the Invention

Overview of Conventional Pipettes:

FIGS. 1-5 show a form of pipette as disclosed in U.S. Patent No. 5,874,296. With reference to FIGS. 1, 2 and 3, an embodiment of a liquid medium exchange apparatus 50 is depicted. The apparatus comprises a pipe 1, a cylinder reservoir 2, a vacuum controller 3, a reservoir control 4 and associated connecting tubing 5, 6, and 7. The pipe 1 has a first end preferably tapered into an opening tip 8, which is used for the drawing and delivery of a liquid medium, a central portion, and a second end comprising an opening 9 for connection to a vacuum source. The second end further provides a convenient handle for an operator to hold the apparatus. The central portion of pipe 1 is configured to cradle or otherwise hold the cylinder reservoir 2 in intimate contact thereto. Additionally, at a forward and a rearward end of the central portion of pipe 1 are located access openings 10 and 11 respectively. Access opening 10 is connected to a corresponding opening 13 in the forward end of the cylinder 2 via tubing 5. The access opening 11 is connected to a corresponding opening 14 in the rearward end of the cylinder 2 via tubing 6. The vacuum controller 3 is disposed between the access opening 11 and the opening 9 of pipe 1. The cylinder 2 has a third opening 15, which is attached to a new liquid source, which is intended to be placed in the culture vessel. Though other means could be used to accomplish the same purpose, the respective heights of each of these openings 13, 14, and 15 in relation to that portion of the cylinder in contact with the pipe 1 perform a useful role in the function of the apparatus. As can be seen in the figures, in the preferred embodiment of the cylinder 2, openings 13 and 15 are placed low in the cylinder, whereas opening 14 is placed substantially higher.

Looking first at FIG. 1, the vacuum controller 3 is seen to comprise a housing 35 suitably configured to receive a seal cap 21. The seal cap 21 provides a convenient on- off means to control the vacuum with which the apparatus 1 is subjected. The seal cap 21 is normally displaced from the housing 35. By causing the seal cap 21 to seal with the housing 35, vacuum can be built up and maintained within the apparatus. Whereas releasing seal cap 21 breaks the vacuum. A configuration of the vacuum controller 3 includes dividing the housing 3 into two individual passages or channels 17 via a dividing wall 16. By adding a sliding partition 18 containing a passage 20 to the seal cap 21, the passage 20 can be made to align or non-align with an internal passage of pipe 1. A biasing means such as spring 19 can be utilized to keep the seal cap 21 in its normally open position.

FIGS. 2 and 3 depict the reservoir control 4 that enables the new liquid medium to be drained from the cylinder reservoir 2. The reservoir control 4 comprises a lever 36 pivotally attached to the pipe 1 by a pin connection 37. A forward portion 22 of the lever 36 is made to overlie the tubing 5. This overlapping forward portion 22 of the lever 36 impinges upon tubing 5 thus pinching off or clamping the tubing 5 between the forward portion 22 and the pipe 1. To assist in this, a biasing means such as a rubber band 23, spring or other suitable mechanism is utilized to keep portion 22 firmly clamped against the tubing 5 thereby eliminating flow. The tubing 5 by necessity must be made sufficiently soft so as to be collapsible upon itself for an embodiment just described to work. Nonetheless, to enable flow through the tubing 5, a rearward portion of the lever 36 ends in a paddle 24 or an equivalent user manipulable button or pad.

Operation of the apparatus 1 is illustrated in FIG. 4. By connecting the device to a "clean" medium container 25 via the tubing 7, which is preferably sterile, and connecting the opening 9 to a waste collecting bottle 26 which is ultimately connected to a vacuum source, the process can begin. The method for transferring the fluids is described as follows:

1) Hold the apparatus 1 with one hand and immerse the opening tip 8 into the old liquid that is intended to be disposed. Flow cannot occur since tubing 5 is clamped closed and the pipe 1 is open to atmosphere through the forward channel 17. Also, the vacuum cannot be utilized since the rearward channel 17 is also open to the atmosphere.

2) Push the seal cap 21 downward until it seats upon the housing 3 thereby causing the passage 20 to align with the inside of the pipe 1. At this point both the pipe 1 and the reservoir 2 are under vacuum. Flow is established through the pipe 1 from the opening tip 8, out opening 9 into the waste collection bottle 26.

3) Simultaneously vacuum is applied to the clean medium container 25 through the tubing 6, the reservoir 2, and the tubing 7 thus filling the reservoir 2 with clean liquid.

4) Under normal circumstances suction is maintained until either one of the following occurs: the seal cap 21 is released, or the opening tip 8 is removed from the liquid or the liquid is completely suctioned from its container.

5) Once the old medium is removed and the reservoir 2 is filled, depressing the paddle 24 causes the forward portion 22 of the lever 36 to pivot about the pin connection 37 thereby unclamping the tubing 5 thus emptying the reservoir 2 into the now emptied container or culture vessel.

The patentee of the '296 patent claims that placement of the opening 14 higher in the cylinder reservoir 2 prevents old medium from entering the reservoir during the vacuum process while also eliminating the inadvertent disposal of the clean liquid unless the reservoir 2 overfills. Other options that may be desirable are to provide graduation markings 12 on the cylinder reservoir 2 so as to accurately control the volume of liquid added to the culture. The '296 patent also teaches that the opening tip 8 can be made to be replaceable, and asserts that this may be desirable if it is required that no cross contamination occur between the old liquid and the new. Additionally two opening tips, as depicted in FIG. 5, could be provided, one for the aspiration process configured as already described and a second tip 60 to which the tube 5 connects. The tube 5 would now be isolated from the first tip 8 and connected directly to the second tip 60. To prevent contamination of the second tip 60 during the aspiration process, the second tip can be vertically coplanar with respect to the first tip but shorter. Unfortunately, the above described structure of the '296 patent is subject to cross contamination because the same conduit is used for new and old medium. Embodiments of the Present Invention:

The present invention incorporates new features and improvements that completely protect against cross-contamination. This can be achieved by employing a second conduit to aspirate old medium, instead of using a first conduit or central chamber, and also employing a movable second conduit to minimize the risk of contact between the aspiration tip of the second, external conduit and the clean medium.

With reference to FIGS. 6, 7, and 11, an embodiment of an improved pipette apparatus 1000 is depicted. The apparatus comprises a first conduit 1004, an external conduit 1010, a body 1002a, and a pipette head assembly 1100 with associated vacuum port 1020, fresh fluid inlet 1101, and fluid removal inlet 1102. The first conduit 1004 has a first end preferably tapered into an opening tip 1006 that is used for the outlet delivery of a liquid medium into container 500. A second end of the first conduit 1004 is directly connected to lower portion 1002b of elongated body 1002a via orifice 1028. Body 1002a and body lower portion 1002b can be comprised of polyethylene in order to provide a tighter fit with eightport manifolds for use in dispensing liquid to 96 wells. Body 1002a and body lower portion 1002b comprise central chamber 1001. The second conduit 1010 comprises a first open end preferably tapered into opening tip 1008 that is used to aspirate material from a container 500. Vacuum port 1020 establishes vacuum conditions to aspirate liquid medium by suction via second conduit 1010 and enable new liquid medium to flow through fresh fluid inlet 1101 into elongated body 1002a.

Pressing control button 1005a of pipette head assembly 1100 causes plunger 1012 to slide downwardly thereby placing seal end 1014 in intimate contact with the internal walls of body lower portion 1002b and sealing orifice 1029. By sealing orifice 1029, replacement medium no longer flows through first conduit 1004 into the culture sample of container 500 during aspiration. Plunger 1012 can be comprised of a solid material to ensure no air leaks occur during negative pressure and thereby allow liquid to flow down central chamber 1001. Plunger 1012 is positioned within central chamber 1001 and head assembly 1100. Body 1002 is in intimate contact with housing ring 1024 of pipette head assembly 1100. The second conduit 1010 has a first end preferably tapered into an opening tip 1008 which is used for inlet of old medium. In one embodiment shown in FIGS. 6, 7 and 11, the second end of second conduit 1010 is in intimate contact with tab 1026 and flexible portion 1009 via orifice 1031. Flexible portion 1009 has a first end in intimate contact with second conduit 1010 and a second end in intimate contact with fluid removal inlet 1102 of vacuum port 1020 via orifice 1030. Vacuum forces within vacuum port 1020 and fluid removal inlet 1102 pull old medium through an external conduit pathway by suction via second conduit 1010, orifice 1031, flexible portion 1009, and orifice 1030 during aspiration. Depressing tab 1026 causes second conduit 1010 and flexible portion 1009 to move downward thereby enabling a user to place opening tip 1008 of second conduit 1010 in the old medium and remove the old medium via a movable pathway external to and below the plane of opening tip 1006 of first conduit 1004, thereby eliminating the risk of cross contamination. It will also be seen that when the user presses control button 1005b, second conduit 1010 is pressed downwardly so that opening tip 1008 of second conduit 1010 is below the level of the tip of first conduit 1004, thereby enabling opening tip 1008 to remove the old medium. In another embodiment depicted in FIG.7, depressing tab 1026 causes second conduit 1010 to move upward or downward within clip 1011 and inlet-outlet clip 1018.

In a further embodiment depicted in FIGS. 8a - 8b, 9, and 10, a user can utilize control button 1005a and control button 1005b to control fluid movement. Referring to FIGS. 8a - 8b and 10, the second conduit 1010 comprises a first open end preferably tapered into opening tip 1008 that is used to aspirate by suction material from a container 500. Also shown is a vacuum port 1023 connectable to a vacuum source, which enables further control of fluid flow. Referring to FIGS. 8a, 9 and 10, control button 1005a controls plunger 1012 and control button 1005b controls second conduit 1010. When the user depresses control button 1005 a, plunger 1012 slides downwardly thereby placing seal end 1014 in intimate contact with the internal walls of body lower portion 1002b and sealing orifice 1029. By sealing orifice 1029, no replacement medium flows through first conduit 1004 into the culture sample of container 500 during aspiration. As shown in FIGS. 8a, 9 and 10, control button 1005b is in intimate contact with the upper portion of second conduit 1010 and spring or rubber mechanism 1005c. In an embodiment shown in FIGS. 8a, 9 and 10, a metallic or rubber spring medium can be used to spring-load control button 1005b. Pressing control button 1005b causes second conduit 1010 to move downward thereby enabling a user to place opening tip 1008 of second conduit 1010 in the old medium and remove the old medium via a movable pathway external to and below the plane of opening tip 1006 of first conduit 1004, thereby eliminating the risk of cross contamination. The second conduit 1010 is thus ready to be placed in the old medium contained in container 500 for removal of the old medium without risk of cross contamination from inadvertent contact between the opening tip 1006 of first conduit 1004 and the old medium. ^• With reference to FIGS. 6 and 7, connection of second conduit 1010 and flexible portion 1009 to body 1002 and first conduit 1004 is depicted, such that inlet-outlet clip 1018 can be used to hold the external inlet conduit 1010 to the first, central conduit (outlet) 1004, wherein second conduit 1010 is in a moveable relationship with the first conduit 1004. Referring to FIGS. 8a and 9, inlet-outlet clip 1018 can be used to hold the external inlet conduit 1010 to the first conduit (outlet) 1004, such that second conduit 1010 is in a moveable relationship with the first conduit 1004. Thus, the external inlet conduit can be slid up and down (either automatically by action of the control button, or manually, by the operator using tab 1026) relative to the first conduit 1004.

With reference to FIGS. 6, 7 and 11, one embodiment of an improved pipette head assembly is depicted in FIG. 11. As shown therein, the external aspect of the head assembly 1100 includes a fresh fluid inlet 1101, a vacuum source port 1020, and a fluid removal inlet 1102 in vacuum communication with the vacuum source port at one end, and second conduit 1010 (inlet) at the other end. The operation of improved pipette apparatus 1000 is shown in FIGS. 6, 7, and 11. By connecting the apparatus to clean liquid medium via tubing and fresh fluid inlet 1101 , as well as connecting the apparatus to a waste-collecting vessel that is ultimately connected to a vacuum source that includes vacuum port 1020, the process can begin.

Referring to FIGS. 8a - 8b, and 9 - 10, another embodiment of an improved pipette head assembly is depicted. As shown therein, the external aspect of a head assembly 1100 comprises a fresh fluid inlet 1101, a vacuum source port 1020, and a vacuum port 1023 in vacuum communication with the vacuum source ports 1020 and 1023 at one end, and second conduit 1010 (inlet) at the other end. The external aspect of a head assembly 1100 further comprises vent 1022. The operation of improved pipette apparatus 1000 is shown in FIGS. 6 - 11. By connecting the apparatus to a clean medium vessel via tubing and fresh fluid inlet 1101, and connecting vacuum port 1023 via tubing to a waste-collecting vessel that is ultimately connected to a vacuum source, the process can begin.

A method for transferring medium using the above described structure is as follows:

1.) After vacuum has been established, a user holds apparatus 1000 with one hand and, in one embodiment, press control button 1005b in order to slide the second conduit 1010 downward and place opening tip 1008 into the old liquid medium held in medium container 500 which is intended to be disposed. In another embodiment, the user manually presses tab 1026 to slide second conduit 1010 downward and place opening tip 1008 into the old medium intended to be disposed. Flow occurs through the second conduit 1010 pathway while control button 1005b or tab 1026 is pressed downward. Flow is maintained and old medium flows out vacuum port 1020 into a waste collection bottle 26. When the desired amount of old medium is removed, opening tip 1008 is moved upward by releasing pressure on control button 1005b or tab 1026.

2.) Simultaneously while aspirating old medium, a user collects replacement medium from replacement medium source 25 via tubing connecting to fresh fluid inlet 1101. No replacement medium flows from first chamber 1001 downward through first conduit 1004 when a user presses control button 1005a. Pressing control button 1005a of pipette head assembly 1100 causes plunger 1012 to slide downwardly thereby placing seal end 1014 in intimate contact with the internal walls of body lower portion 1002b and sealing orifice 1029. By sealing orifice 1029, no replacement medium flows through first conduit 1004 during aspiration. 3.) Release of replacement medium from the central chamber via the first conduit 1004 into medium container 500 is effectuated by releasing control button 1005a, thereby withdrawing the plunger 1012, breaking the seal between the internal walls of lower portion 1002b and allowing the replacement medium to flow through first conduit 1004, out opening tip 1006 and into medium container 500. Release of replacement medium can be stopped at any time by pressing control button 1005a partially downward thereby re-creating a seal with seal end 1014 and the internal walls of lower portion 1002b.

Those skilled in the art will appreciate that various changes can be made in the above-described structure and method steps, and the various changes are still within the scope of the invention as defined by the following claims.

Claims

ClaimsWhat is claimed is:

1. A liquid medium exchange device comprising: a first conduit having a tip for delivery of medium to the container, and an opposed, open end operable to be connected to a vacuum source and a medium source; a second conduit having a tip for withdrawal of material from the container and an opposed, open end operable to be connected to a vacuum source; support elements for said first and second conduits, said support elements are configured to mount said first and second conduits such that said tip of said second conduit is movable relative to said tip of said first conduit; and first and second control elements wherein said first control element is integrated in combination with said open end of said first conduit and operative to activate said first conduit for delivery of material from the material source to the container, and said second control element is integrated in combination with said open end of said second conduit and operative to move said second conduit relative to said first conduit wherein material is withdrawn from the container by said second conduit utilizing the vacuum source.

2. The device of claim 1, wherein actuation of said first control element enables control of the medium flow.

3. The device of claim 1, wherein actuation of said second control element enables withdrawal of the medium.

4. The device of claim 1, wherein said second control element comprises a tab configured for integration with said second conduit proximal to said tip of said second conduit.

5. The device of claim 1, wherein said second conduit further comprises a flexible portion.

6. The device of claim 5, wherein the flexible portion comprises a metallic spring element.

7. The device of claim 5, wherein the flexible portion comprises a rubber spring element.

8. The device of claim 1 further comprising: a head assembly; an elongated body connected to said head assembly, said elongated body including a chamber; and a plunger configured for sliding movement within said head assembly and said chamber.

9. The device of claim 8, wherein said elongated body is comprised of polyethylene.

10. The device of claim 8, wherein said plunger is comprised of a solid material.

11. The device of claim 8, wherein said first control element comprises a button configured for integration with said head assembly.

12. A method of medium withdrawal, the method comprising: establishing a vacuum; actuating a first control element to prevent the flow of a medium through a first conduit; and actuating a second control element to slide a tip of a second conduit, wherein said tip moves relative to a tip of a first conduit; and withdrawing the medium.

13. A method of medium delivery, the method comprising: establishing a vacuum; and actuating a first conduit control element to allow the flow of a medium through a first conduit to dispense said medium.