WO2009088384A1

WO2009088384A1 - Controlled flow rate micro-pipettes

Info

Publication number: WO2009088384A1
Application number: PCT/US2008/000219
Authority: WO
Inventors: Steen M. Willadsen
Original assignee: Willadsen Steen M
Priority date: 2008-01-07
Filing date: 2008-01-07
Publication date: 2009-07-16

Abstract

Micro-pipettes featuring a flow resistance device, preferably in the form of a section, preferably close behind the distal tip of the pipette, where the diameter of the lumen has been drastically reduced to slow down the flow rate of fluid in and out of the pipette The design allows the flow rate to be precisely controlled, irrespective of other design and operational features of the pipette, provided that it is operated with a fluid column extending beyond the constriction, into the shaft of the pipette The flow of fluid in and out of micro-pipettes of this design is easily and reliably controlled pneumatically with the aid of any of the suction/ injection devices currently used for operating micro-pipettes in micromanipulation of cells and tissues, even an ordinary disposable plastic syringe

Description

Patent Application of

Steen M. Willadsen

for

TTTLE: CONTROLLED FLOW RATE MICRO-PIPETTES

CROSS REFERENCE TO RELATED APPLICATIONS: Provisional Application mailed AUG 2^nd 2006

FEDERALLY SPONSORED RESEARCH: None

SEQUENCE LISTING: None

BACKGROUND OF THE INVENTION

This invention relates to instruments used in micromanipulations involving cells, subcellular fragments, and elements isolated from, or to be incorporated into, cells.

Micromanipulations of cell- and tissue samples play a central role in modern biology, both at the experimental and the applied level. Gene injection, cytoplasmic and nuclear transplantation, intra-cytoplasmic sperm injection, polar body- and embryo biopsy, fragment removal, and assisted embryo hatching, blastomere separation, blastomere aggregation and blastocyst injection for production of chimaeric embryos may serve as examples of the range of procedures that are currently in widespread use just in the area of assisted human reproduction, mammalian embryology generally, and stem cell science.

A wide range of specially designed microsurgical instruments (hence termed "micro-tools") have been invented and developed to facilitate the various and varied procedures that comprise micromanipulation of cells and tissues. Most of these instruments take the form of micro-injection needles or micro-pipettes (hence collectively termed "micro-pipettes"). Glass capillary tubes (usually with an outer diameter of ~lmm) are used as the starting material for production of micro-tools. A section of the glass tube is heated and drawn to a tapering needle with the aid of an programmable pipette-puller. The heating and pulling parameters are adjusted so that the desired length and degree of tapering of the pulled section of the resulting straight hollow needle are obtained. The precise shape and dimensions of the micro-tool tip depend on the particular operation for which it is intended, and are critical for its effective specific use. Micro-forges and micro-grinders are used to fashion the tips of the micro-tools to the correct shape and dimensions. The general design of the most commonly used types of micro-tools is shown in Rg.1.

For the shaft of the micro-tool, the original diameter of the capillary tube is retained, so that the proximal end of the micro-tool may be fitted air tight into the distal end of the tubular metal instrument holder of a micromanipulator. The micromanipulator is a mechanical device designed to scale down the hand- and finger movements of the operator to the level required for the particular procedure. With the aid of the micromanipulator precise control over the movements of the micro-tool in three-dimensional space right down to the micron level is achieved. In the case of micro-pipettes, a thin flexible tube is fitted air tight into the opposite end of the instrument holder and connected to a suction/ injection device the purpose of which is to control the flow of fluid into and out of the micro-pipette.

Precise control of the flow, in and out of the micro-pipette, of fluid and any particulate material suspended therein (e.g. cells, cell nudei or cytoplasm fragments) is essential for the effective and successful use of the pipette. The ideal is a readily controllable rate of flow which allows the appropriate, miniscule, volume to be delivered precisely at its intended destination, and which may be stopped at will at any time and will then remain arrested so that the micro- tool can left "in neutral", i.e. without any suction or expulsion occurring unintentionally. The latter feature will allow the operator to safely free a hand for other tasks as the need arises, without having to worry about the micro-pipette causing havoc or becoming incapacitated.

However, the conventional micro-pipette design does not in itself ensure realization of this ideal. For with the conventional design the main considerations are:

1) that the tip of the micro-pipette (including, in particular, the bore and outer diameter of the tip) must meet the specific requirements of its intended use,

2) that the shaft of the micro-pipette must fit into the instrument holder,

and

3) that the instrument is shaped/ curved in such a way that its tip section can be positioned properly in the field of operation on the miσoscopic stage.

The quantities of medium and bulk of material to be handled and moved about are miniscule, but vary between different procedures. This is reflected in the tip bore of the pipettes used. For handling whole denuded eggs or large blastomeres, for instance, a pipette with a tip bore of ~ 100 micrometer might be needed, whereas a nudear transfer pipette might have a tip bore of less than one tenth of that. Conventional micro-pipettes of different tip bore have widely different basic operating characteristics. Generally, the wider the tip bore of the micro-pipette, the more difficult it is to control the flow rate. But even micro-pipettes with tip bore of 10 micrometer or less require a high level of skill and experience to operate efficiently.

Control of flow in and out of micro-pipettes is established and maintained primarily with the aid of suction/ injection devices. The latter are of varied design and sophistication, ranging from ordinary disposable plastic syringes, which are relatively inexpensive, but also very difficult to use with conventional micro-pipettes for anyone except a very highly trained and experienced operator, to complex microinjection systems which are somewhat easier to use, but still demand a fairly high level of skill and experience to operate, and also require maintenance expertise to keep in order. In addition, these sophisticated systems are expensive.

Even with the most sophisticated suction/ injection devices available and dose attention and care on the part of the operator, control of flow rate is tricky to establish, and is readily lost while the micropipette is in use, especially if the system is operated with air in the lines - "pneumatically". Loss of control in turn can, and all too often does, lead to loss or destruction of valuable, even irreplaceable biological material. This can easily occur, particularly if the operator allows his or her attention to stray, or even briefly removes the controlling hand from the suction/ injection device, to attend to a different element or aspect of the procedure under execution.

If the micropipette is to be operated pneumatically, experience has taught that it is essential to keep the fluid in the thin part of the micro-pipette. Only in this way is it possible to establish sufficient control over the flow rate. However, the "control" thus established is precariously balanced between a number of different forces and factors in complex interaction. In practice control cannot be maintained if the fluid rises into the wider part of the micro-pipette. This could easily happen by capillary effect. Hence control measures must be actively maintained even in intervals during the procedure when the micro-pipette is not being actively used.

Further complications are caused by tiny air bubbles that tend to form at the boundary between liquid and air as it moves up the thin section of the micro-pipette during suction. These air bubbles easily get trapped in the tapering tip section of the micro-pipette when the direction of flow is turned. The result is that the micro-pipette is wholly or partially blocked and responds erratically, filling and emptying with unpredictable speed. This leads to further disruption of function, and ensuing havoc, and frustration on the part of the operator, who is then more likely to make mistakes, even when operation is resumed with fresh instruments.

If it is attempted to operate the micro-pipette "pneumatically" with the fluid column beyond the point of capillary equilibrium, i.e. reaching the relatively wide shaft of the pipette, where boundary effects and air bubbles are less likely to cause unmanageable complications, the rate of flow will be far too fast to control the movements of the minute quantities that have to be dealt with.

The problems mentioned above can be avoided, or reduced, by using a biologically inert, viscous, essentially non-compressible fluid in the system instead of, or in combination with, air. Mineral oil is the most common choice. However, such "hydraulic" operation brings in another set of problems, that are primarily due to differences in viscosity and glass adhesion between the water-based cell-manipulation medium and the "inert" hydraulic fluid, and molecular boundary phenomena between the two types of fluid and the glass wall. The result very often is that droplets of manipulation medium and/ or of hydraulic fluid are separated out from the main columns. The droplets get mixed helter-skelter along the thin section of the micro-pipette, trap cells and other particulate matter, and stick to the wall of the micro-pipette, so that it becomes ineffective or completely useless. All of the problems described or alluded to above can be effectively and reliably solved by the use of the controlled flow rate micro-pipettes whjch constitute the present invention. Controlled flow rate pipettes feature a flow resistance device. In the following the terms "flow resistance device" and "constriction" are used interchangeably about any device whereby the effective diameter of any section of a tubular instrument is reduced.

The idea behind the present invention - of addressing the control of flow rate specifically and separately in the design of the micro-pipette itself - has not, to the knowledge of this author, been described in the literature dealing with cell manipulations, including current assisted reproductive technology or mammalian embryology manuals. Nor is the author aware of any laboratory, other than his own, where the idea has been conceived or successfully applied, even though many hundreds of dinical and experimental laboratories are in existence where cell manipulations of the types with which the invention is concerned are fully integrated in the daily routine. The invention cannot therefore be considered to have been obvious to experts.

All of the laboratories mentioned above, and indeed most others in which micromanipulations of cells are used, stand to benefit substantially from the present invention. The invention is therefore useful.

SUMMARY

The invention takes the form of a range of micro-pipettes featuring a flow resistance device - "a constriction". The specific purpose of the flow resistance device is to enable an operator to control precisely and reliably the rate of flow in and out of the pipette, irrespective of other specific design features related to the intended use of the pipette.

In the preferred embodiments of the invention, the flow resistance device/ constriction takes the form of a section of the micro-pipette where the effective diameter of the lumen has been drastically reduced compared to the tip bore of the pipette.

The term "controlled flow rate micro-pipettes" is used collectively about the whole range of pipettes featuring a constriction or an equivalent flow resistance device. The range of controlled flow rate micropipettes includes, but is not limited to:

1. Pipettes for holding and handling eggs and embryos

2. Pipettes for embryo biopsy

3. Pipettes for handling blastomeres

4. Pipettes for intra-cytoplasmic injection of sperm

5. Pipettes for extracting, handling, and/ or transfer of cell nuclei or cytoplasm

6. Pipettes for assisted hatching

7. Pipettes for insertion of cells into the cavity of blastocysts

8. Pipettes for enucleation of eggs, zygotes and/ or blastomeres

9. Pipettes for removal of cytoplasmic fragments

10. Pipettes for handling of somatic cells

11. Pipettes for handling of embryonic stem cells

In controlled flow rate micropipettes, the constriction is preferably placed near the distal end of the pipette.

As long as the column of fluid extends beyond the constriction and into the relatively wide shaft of the micro-pipette, and preferably slightly above the fluid level that would result from capillary effects under the relevant operating circumstances, the flow in and out of the pipette in response to any change in the pressure applied at the proximal end of the shaft is drastically reduced. The flow rate is hereby slowed down sufficiently for the micro-pipette to be operated pneumatically, without loss of control, with any of the suction/ injection devices currently used, including, significantly, even an ordinary disposable syringe. The resistance to flow caused by the constriction also reduces flow in and out of the micro- pipette to insignificant levels when the application of pressure or suction at the proximal end of the pipette is discontinued, allowing the micro-tool to be left unattended "in neutral" for brief periods of time.

The degree of control obtained depends primarily on the diameter of the constriction. Since the degree of control sought depends on the specific use for which the particular micro-pipette is intended, the precise diameter of the constriction may be adjusted accordingly.

While it is possible to adapt the invention to control of the flow of fluid in and out of the pipette indirectly through control of air-flow established with an air flow resistance device, pipettes of such design are less convenient to operate than the ones described in detail here.

The principle of the invention is useful for producing other types of controlled flow rate pipettes than the micro-pipettes described in detail here.

The observations leading to the invention are supported by Poiseuille's law concerning the flow rate of a fluid under pressure, in a tube.

where

Δ Vl Δ t is the volume units flowing per time unit Pl - P2 is the pressure difference between the two ends of the tube, R is the radius of the tube L is the length of the tube Ti is the coefficient of viscosity

According to this, a reduction of the diameter of a section of a tube to e.g. one fifth, everything else equal, should reduce the flow rate through that section to 1/625 of its previous value. Poiseuille's law does not apply unreservedly under the operating conditions of micro-pipettes. Nevertheless, this simple calculation gives some idea of the flow rate reduction achievable by reducing the diameter of a section of the tube. And because of the reduction in the flow rate, control is readily established even when relatively high positive or negative pressures are applied at the proximal end of the shaft of the micro-tool.

Advantages of the invention:

The invention made by the author has made it much more easy to establish and maintain precise control over the flow of fluid and suspended particulate matter in and out of micro- pipettes, irrespective of bore and other characteristics of other parts of the pipette.

Trials with inexperienced operators have demonstrated that the skill needed to control the flow of fluid and particulate matter in and out of a controlled flow rate micro-pipette can be acquired with relatively little practice. But more importantly, an operator already skilled in the use of micro-pipettes of conventional design will immediately realize that controlled flow rate micro- pipettes represent a very significant improvement with regard to precision, reliability, and ease of operation.

A crucial advantage of the invention is that it allows the problems of flow rate control to be effectively solved separately without interference with other design and operational features of the micro-pipette.

A further advantage of the constriction placed as in the preferred embodiments of the invention is that should a cell or membrane bounded cell fragment be sucked far enough into the pipette to reach it, that cell or cell fragment will simply be detained at the distal end of the constriction, and not sucked out of view and into the more proximal parts of the pipette, where it might easily get lost or destroyed.

A further advantage of the invention is that it allows the use of a disposable syringe as suction/ injection device. This improves the possibilities for establishing and maintaining aseptic conditions during cell manipulations.

Still further advantages will become apparent from consideration of the ensuing description and drawings. DRAWINGS

Fig. 1. is a representation of the most commonly used general design of micro-pipettes for cell micromanipulation as applied in assisted human reproduction and mammalian embryology. The dimensions, the number and degree of bendings, as well as the form and design of the tapering tip section of the pipette varies according to intended use.

Fig. 2 is a schematic enlarged representation of the tip section of a controlled flow rate micro-pipette with the flow resistance device (the constriction) situated in the distal portion of the bend of the tip section of the instrument, but without interfering restrictively with the other design features and operating dimensions of micro-pipette.

RG. 3 shows an example of an egg- or embryo holding pipette with a constriction incorporated.

RG. 4 shows an example of a blastomere biopsy and -handling pipette with a constriction incorporated.

RG. 5 shows an ICSI(Intra Cytoplasmic Sperm Injection) pipette with a constriction incorporated.

REFERENCE NUMERALS For Figs. 1-5:

1. shaft

2. pulled section = thin part

3. proximal end

4. distal end = tip

5. bend

6. tip section

7. flow resistance device = constriction

8. tip bore DETAILED DESCRIPTION

The preferred embodiment of the invention is a micro-pipette made of glass featuring a constriction.

In the preferred embodiment of the invention, the constriction 7 takes the form of a section of the micro-pipette where the internal diameter has been drastically reduced by local thickening, (heat-induced compaction) of the glass wall of the micro-pipette.

The constriction may be placed anywhere along the length of the micro-pipette (Rg. 1) as long as the most distal tip section 6 of the pipette is left as originally designed for its particular use.

Note that in preferred embodiments of the invention, the constriction 7 is placed in the thin part of the micro-pipette, just behind the distal tip section 6, starting immediately before, and extending into, the (usually about 30 °) bend 5 that is a design feature of most micro-tools currently used (cf. Rg. 1).

In conventional micro-pipettes the bend 5 is produced by controlled localized heat whereby the part of the originally straight pulled part of the capillary tube nearest to the heating filament of the micro-forge is softened to the point where it bends, until the desired angle is obtained. Often slight compaction of the glass wall occurs on the side towards which the tube is bent. However, in conventional pipettes, the thickening of the wall is almost imperceptible, and insignificant in the context of controlling flow rate. Indeed, substantial thickening of the wall in a conventional pipette would generally be considered evidence of poor craftsmanship on the part of the toolmaker.

In the preferred embodiment of the invention, the constriction 7 is conveniently produced in the process of producing the bend 5, by first exaggerating the bending in one direction and then repeating the process, but in the opposite direction, until sufficient compaction of the glass has occurred to reduce the lumen of that section of the tube to the desired diameter. At that point, the bend 5 is adjusted to the desired angle if necessary (or straightened out, if the micro-pipette is required to be straight).

The presence or absence of one or several bends in the particular micro-pipette is irrelevant to the effectiveness of the invention for flow rate control. However, the possibility of insertion of more than one constriction has opened design options for micro-pipettes with additional operational advantages for specific uses.

The degree of reduction of the lumen of a section of the micro-pipette necessary to establish optimal control over the operation of a particular type of micro-pipette varies from about 2 micrometer, or even slightly wider, adequate for most egg or embryo holding pipettes ( Rg. 3) down to 0.5 micrometer or less for blastomere biopsy pipettes (Fig. 4) and ICSI needles (Fig. 5).

In the preferred embodiment of the invention, the length of the constriction 7 varies from approximately a hundred micrometer in ICSI needles (Fig. 5) to several hundred micrometer in egg and embryo holding pipettes (Fig. 3). Generally, in the process of producing the desired narrowness of the constriction 7 an appropriate length of the constriction also results.

OPERATION

Controlled flow rate micro-pipettes may be operated efficiently and reliably either pneumatically or hydraulically with any suction/ injection device from the whole range currently being used with the corresponding conventional micropipettes. Significantly, even an ordinary disposable plastic syringe will suffice.

Prior to operating a controlled flow rate micro-pipette, it is important to ensure that it is filled with fluid well beyond the constriction 7, so that during operation fluid only, and not air, flows through the constriction.

Furthermore, while most conventional micro-pipettes become practically impossible to operate pneumatically when the column of manipulation medium is allowed to fill or extend beyond the relatively narrow distal part 2 of the pipette, it is important for the optimal functioning of controlled flow rate micro-pipettes intended for precise movement of minimal volumes, such as biopsy pipettes and ICSI needles, that the fluid column reaches a point slightly beyond the level of capillary equilibrium under the circumstances. Hereby is also ensured, that air bubbles will not interfere with the flow of fluid through the constriction 7. The reservoir of fluid proximally to the constriction 7 is also useful to have for rinsing the tip section 6 of the pipette if the latter threatens to become blocked in the course of the micromanipulation procedure.

Claims

CLAIMS: I claim:

1. A tubular instrument into which fluid may be drawn and subsequently dispensed, featuring a flow resistance device.

2. A tubular instrument as described under 1. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

3. A tubular instrument into which fluid may be drawn and subsequently dispensed, featuring a flow resistance device which allows the flow of fluid into and out of the instrument to be controlled.

4. A tubular instrument as described under 3. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

5. A tubular instrument into which fluid may be drawn and subsequently dispensed, featuring a flow resistance device which allows the flow of fluid into and out of the instrument to be controlled independently of other design and operational features of the instrument.

6. A tubular instrument as described under 5. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

7. A micro-pipette featuring a flow resistance device.

8. A micropipette as described under 7. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

9. A micro-pipette featuring a flow resistance device which allows the flow of fluid in to and out of the pipette to be controlled.

10. A micro-pipette as described under 9. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

11. A micro-pipette featuring a flow resistance device which allows the flow of fluid into and out of the pipette to be controlled independently of other design and operational features of the pipette.

12. A micro-pipette as described under 11. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

13. A tubular instrument for micromanipulation of tissues, cells, or subcellular components, into which fluid may be drawn and subsequently dispensed, featuring a flow resistance device.

14. A tubular instrument as described under 13. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

15. A tubular instrument for micromanipulation of tissues, cells, or subcellular components, into which fluid may be drawn and subsequently dispensed, featuring a flow resistance device which allows the flow of fluid into and out of the instrument to be controlled.

16. A tubular instrument as described under 15. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening.

17. A micro-pipette for micromanipulation of tissues, cells, or subcellular components, featuring a flow resistance device.

18. A micro-pipette as described under 17. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

19. A micro-pipette for micromanipulation of tissues, cells, or subcellular components, featuring a flow resistance device which allows the flow of fluid into and out of the pipette to be controlled.

20. A micro-pipette as described under 19. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

21. A micro-pipette featuring a flow resistance device which allows the flow of fluid into and out of the pipette to be controlled independently of other design and operational features of the pipette.

22. A micro-pipette as described under 21. in which the flow resistance device takes the form of a section of any length where the diameter of the lumen is less than half the diameter of the distal tip opening of the tool.

23. A flow resistance device incorporated in the design of a tubular cell manipulation tool for the purpose of controlling the rate of flow in and out of the tool.

24. A flow resistance device as described under 23 situated between 0.05 mm and 100.0 mm from the distal tip opening of a cell manipulation tool.

25. A micro-pipette for micromanipulation of tissues, cells, or subcellular components, any section of which has a lumen diameter of less than half the diameter of the distal tip opening of the pipette.