U.S. patent application number 09/836280 was filed with the patent office on 2001-10-18 for method and apparatus for making density gradients.
Invention is credited to Anderson, Norman G..
Application Number | 20010031688 09/836280 |
Document ID | / |
Family ID | 24200757 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031688 |
Kind Code |
A1 |
Anderson, Norman G. |
October 18, 2001 |
Method and apparatus for making density gradients
Abstract
A float is used for preparing a density gradient in a
parallel-walled vessel. The float has an outer peripheral surface
that has a diameter smaller than an inner diameter of an inner
surface of the vessel. With the float placed inside the vessel a
liquid is introduced onto the float such that the liquid flows
around the float between the float and the inner wall of the
vessel. The shape and configuration of the float slows the velocity
of the liquid such that there is only laminar flow as the liquid
contacts other liquid below the float. Elimination of turbulent
flow prevents mixing of different liquid introduced into the same
vessel thereby forming layers of fluid. Preferably, the vessel is a
centrifuge tube. In one embodiment, the outer diameter of the float
is large enough to cause capillary action between the float and the
inner surface of the centrifuge tube to force liquid to remain
between the float and the inner surface of the centrifuge tube.
Inventors: |
Anderson, Norman G.;
(Rockville, MD) |
Correspondence
Address: |
Dean H. Nakamura
Roylance Abrams Berdo & Goodman
1300 19th Street N.W.
Washington
DC
20036
US
|
Family ID: |
24200757 |
Appl. No.: |
09/836280 |
Filed: |
April 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09836280 |
Apr 18, 2001 |
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09551314 |
Apr 18, 2000 |
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Current U.S.
Class: |
494/37 ; 141/100;
220/216; 494/85 |
Current CPC
Class: |
B01L 99/00 20130101;
B01L 3/50215 20130101; G01N 15/042 20130101; G01N 33/491
20130101 |
Class at
Publication: |
494/37 ; 494/85;
141/100; 220/216 |
International
Class: |
B04B 011/00 |
Claims
What is claimed is:
1. A method for producing liquid density gradients in a vessel
using a float within the vessel, the method comprising the steps
of: inserting the float in the vessel; introducing a first liquid
into the vessel; and introducing a second liquid into the vessel
such that the second liquid contacts at least one surface of the
float upon entering the vessel, contact between surfaces of the
float and the second liquid allowing the second liquid to form a
layer above the first liquid thereby forming separate layers of
liquid.
2. The method as set forth in claim 1 further comprising:
introducing a third liquid into the vessel such that the third
liquid contacts at least one surface of the float upon entering the
vessel, contact between surfaces of the float and the third liquid
allowing the third liquid to form a third layer above the second
liquid thereby forming three separate layers of liquid.
3. The method as set forth in claim 2 further comprising:
introducing a fourth liquid into the vessel such that the fourth
liquid contacts at least one surface of the float upon entering the
vessel, contact between surfaces of the float and the fourth liquid
allowing the fourth liquid to form a fourth layer above the second
liquid thereby forming four separate layers of liquid.
4. The method as set forth in claim 2 wherein the first liquid has
a density greater than the density of the second and third liquids
and the density of the second liquid is greater than the third
liquid.
5. The method as set forth in claim 1 wherein in said insertion
step, the float is surrounded by an inner surface of the vessel
such that during subsequent steps, the various liquids undergo
capillary action contacting both an outer peripheral surface of the
float and the inner surface of the vessel as the fluid is drawn by
gravity under the float.
6. An apparatus for producing liquid density gradients, the
apparatus comprising: a vessel; and a float positionable in said
vessel, said float being formed with at least one surface that is
shaped to inhibit acceleration of fluid introduced into said vessel
thereby restricting turbulent flow of the fluid.
7. The apparatus as set forth in claim 6, wherein an outer
peripheral surface of said float has a shape conforming to an inner
surface of said vessel.
8. The apparatus as set forth in claim 7 wherein said vessel and
said outer peripheral surface of said float have a round shape.
9. The apparatus as set forth in claim 7 wherein said vessel and
said outer peripheral surface of said float have a generally square
shape.
10. The apparatus as set forth in claim 7 wherein said vessel and
said outer peripheral surface of said float have a triangular
shape.
11. The apparatus as set forth in claim 7 wherein said outer
peripheral surface of said float and said inner surface of said
vessel are sized such that in response to fluid being introduced
into said vessel above said float, the fluid undergoes capillary
action moving downward beneath said float in said vessel.
12. The apparatus as set forth in claim 6, wherein said vessel is a
centrifuge tube.
13. A float for use in preparing a density gradient in a
parallel-walled vessel wherein said float has an outer peripheral
surface having a diameter smaller than an inner diameter of an
inner surface of said vessel, and, with said float placed into said
vessel and in response to liquid being placed onto said float the
liquid flows around said float between said float and said inner
wall of said vessel.
14. A float as set forth in claim 13, wherein said vessel is a
centrifuge tube.
15. A float as set forth in claim 14 wherein said outer diameter of
said float is large enough to cause capillary action between said
float and said inner surface of said centrifuge tube to force
liquid to remain between said float and said inner surface of said
centrifuge tube.
16. A float as set forth in claim 14 wherein said float has a
conical upper surface which tapers down from a central apex to an
upper edge of said outer peripheral surface.
17. A float as set forth in claim 16 wherein said outer peripheral
surface has a lower edge and said float has a lower surface that
tapers downward from said lower edge to a central portion
thereof.
18. A float as set forth in claim 17 wherein said upper edge and
said lower edge are rounded over.
19. A float as set forth in claim 17 wherein said upper edge has a
sharp edge.
20. A float as set forth in claim 17 wherein said central portion
of said lower surface includes a pointed region.
21. A float as set forth in claim 16 further comprising a pin fixed
to said float at said central apex.
22. A float as set forth in claim 13 wherein said float comprises a
plastic material having a density less than water such that said
float floats on a water based solution.
23. A float as set forth in claim 13 wherein said float comprises
polypropylene.
24. A float as set forth in claim 13 wherein said float comprises
polyethylene.
25. A float as set forth in claim 13 wherein said float is formed
with a central cavity having a volume sufficient to reduce the
buoyant density of said float to less than that of water.
26. A method for preparing a gradient in a vessel, comprises the
steps of: inserting the float of claim 13 into the vessel;
introducing a liquid into the vessel above the float such that the
liquid flows around the float into a portion of the vessel under
the float.
27. A method as set forth in claim 26, wherein the float is
inserted into the vessel prior to addition of any fluids to the
vessel.
28. A method as set forth in claim 26, comprising the step of:
introducing a first liquid into the vessel before inserting the
float into the vessel, the first liquid forming a first layer of
the gradient and the liquid introduce in the second step in claim
26 forming a second layer of the gradient.
29. A method as set forth in claim 26, wherein the gradient a
continuous gradient.
30. A method as set forth in claim 26, wherein the gradient
comprises a plurality of layers of liquid, each liquid having
decreasing densities.
31. A method as set forth in claim 29, further comprising the steps
of: introducing a second liquid into the vessel above the float
such that the second liquid flows around the float into a portion
of the vessel under the float and above the previously introduced
liquid; introducing a third liquid into the vessel above the float
such that the third liquid flows around the float into a portion of
the vessel under the float and above the previously introduced
second liquid; and allowing the vessel to sit a predetermined time
interval sufficient to allow partial diffusion of the layers of
liquid to convert a step gradient into a semi-continuous
gradient.
32. An apparatus for isolating nucleated cells from blood
comprising: (a) a vessel containing a blood cell separating medium;
and (b) a float positionable in said vessel, said float being
formed with at least one surface that is shaped to inhibit
acceleration of a blood sample introduced into said vessel, thereby
restricting turbulent low of said blood sample onto said separating
medium.
33. The apparatus of claim 32, wherein said medium comprises
silica.
34. The apparatus of claim 32, wherein said medium comprises a
sugar.
35. The apparatus of claim 32, wherein said medium comprises
ficoll.
36. An apparatus for isolating nucleated cells from a blood sample
comprising: (a) a vessel containing a blood cell separating medium;
and (b) the float of any one of claims 13-25, wherein said liquid
is a blood sample.
37. A method for isolating nucleated cells from blood comprising:
(a) introducing a blood sample into the apparatus of claim 32; and
(b) centrifuging said sample in said vessel to produce a gradient,
wherein nucleated cells separate and form a discrete layer in said
gradient.
Description
BACKGROUND OF THE INVENTION
[0001] A. Field of the Invention
[0002] The present invention relates to an apparatus and method for
making a multiple density layers or gradients of fluid in a vessel
in a highly reproducible manner using a float that floats on the
surface of the fluid within the vessel.
[0003] B. Description of the Related Art
[0004] There are various fields where it is desirable to have
density layers or gradients of fluid within a vessel for such
purposes as the separation of matter, determining density, etc.
Such density layers include, for example, a solution retained in a
vessel where the fluid is divided into a plurality of layers, each
layer having differing concentrations of a soluble material or
solute. For example, a bottom or first layer of fluid may have a
concentration of a solute that is X moles per liter; a second layer
immediately above the first layer may have a concentration of 0.8 X
moles per liter; a third layer above the second layer may have a
concentration of 0.6 X moles per liter; and a fourth layer having a
concentration of 0.4 X moles per liter.
[0005] Liquids having gradients of temperature, concentration,
density and color have been previously prepared. Liquid density
gradients have been used for many years, for a wide variety of
purposes, in a number of different industries. The inventor has
numerous publications and patents regarding certain aspects of
gradient formation and use including Anderson, N. G. Mechanical
device for producing density gradients in liquids. Rev. Sci. Instr.
26: 891-892, 1955; Anderson, N. G., Bond, H. E., and Canning, R. E.
Analytical techniques for cell fractions. I. Simplified gradient
elution programming. Anal. Biochem. 3: 472-478, 1962; Anderson, N.
G., and Rutenberg, E. Analytical techniques for cell fractions. A
simple gradient-forming apparatus. Anal. Biochem. 21: 259-265,
1967; Candler, E. L., Nunley, C. E., and Anderson, N. G. Analytical
techniques for cell fractions. VI. Multiple gradient-distributing
rotor (B-XXI). Anal. Biochem. 21: 253-258, 1967.
[0006] A variety of other methods for making density gradients have
been developed, and Bock, R. M. and Ling, N.-S., Anal. Chem. 26,
1543, 1954, and Morris, C. J. O. R, and Morris, P., Separation
Methods in Biochemistry, Pitman Publishing, 2nd ed. (1976) have
reviewed many of these. Only one of these methods allowed gradients
to be made from multiple solutions, each having a different
combination of reagents (Anderson, et al, "Analytical Techniques
for Cell Fractions. I. Simplified Gradient Elution Programming",
Analytical Biochemistry 3: 472-478, 1962) More recent innovations
include the use of pumps and pistons, which are differentially
controlled by microprocessors, e.g., the Angelique gradient maker
(Large Scale Proteomics Corp. Rockville, Md.). Gradients may also
be generated during high speed centrifugation by sedimenting a
gradient solute such as cesium chloride or an iodinated x-ray
contrast medium such as iodixanol. Gradients may be initially
prepared as step gradients and linearized by diffusion, by gentle
mixing, or by freezing and thawing. A list of references covering
existing methods follows.
[0007] Density gradients are used to make two basic types of
separations. The first separates particles on the basis of
sedimentation rate (rate-zonal centrifugation), in which case
particles are separated on the basis of the size and density (and
to a lesser extent shape) and particles will sediment farther if
centrifuged for a longer period of time. The second separates
particles on the basis of isopycnic banding density, in which case
particles reach their equilibrium density level, and do not
sediment farther with continued centrifugation.
[0008] Four types of gradients are in general use with either of
these basic methods. The first includes step gradients, made by
layering a series of solutions of decreasing density (if the
solutions are introduced one above the other), and of increasing
density (if the solutions are introduced sequentially to the bottom
of the tube). The second type comprises linear continuous gradients
usually made by a mechanical gradient maker. These are usually
introduced slowly through small tubing to the bottom of the
centrifuge tube. Linear gradients for either rate zonal or
isopycnic zonal centrifugation are useful for resolving very
heterogeneous mixtures of particles.
[0009] The third type of gradient is non-linear, and may be
designed to separate particles having a very wide range of sizes or
densities. Non-linear gradient may be designed to separate
particles on the basis of both sedimentation rate and isopycnic
banding density in the same gradient, in which case some particles
reach their isopycnic level at some point in the gradient, while
others are still sedimenting. Generally such combined separations
involve larger and denser particles which band near the bottom of
the gradient, while other smaller, and usually lighter particles
are still sedimenting in the upper portion of the gradient.
[0010] The fourth type of gradient is generated in a high
centrifugal field by sedimentation of the major gradient solute,
and is usually used for isopycnic banding.
[0011] Many reasons exist for desiring to control gradient shape.
Gradient capacity (i.e., the mass of particles which can exist in a
zone without causing a density inversion) is a function of gradient
slope, and a steep gradient can support a greater mass of particles
per unit gradient length than can shallow gradients. The greatest
particle mass concentration in a gradient separation usually occurs
immediately beneath the sample zone shortly after centrifugation is
started. As different particles separate in the length of the
gradient, the possibility of an overloaded zone diminishes. For
this reason it is desirable to have a short steep gradient section
immediately under the sample zone, where the highest gradient
capacity is required.
[0012] An additional reason for desiring to control gradient shape
is that when a population of particles is present that differ
little in sedimentation rate, these can best be separated by
sedimentation through a longer shallower section of the gradient.
Such shallow sections are usually near the center of a
gradient.
[0013] In the majority of density gradient separations, the
gradients and their chemical composition are designed to optimize
the separation of one or a few particles types. This accounts for
the very large number of different gradient recipes that have been
published for subcellular fractionation. Those used for the
isolation of mitochondria, for example, are usually quite different
from those used to isolate nuclei. For example, traces of divalent
cations are required to control nuclear swelling, whereas such ions
are generally deleterious to other subcellular particles. Low
concentrations of nonionic detergents remove cytoplasmic
contamination from nuclei, but are deleterious to the endoplasmic
reticulum. Hence there has been no one procedure or gradient that
has been optimized for the systematic separation of the majority of
all subcellular particles. There is a need for reproducible means
for including in gradients zones containing salts, detergents,
enzymes and other reactive substances that would increase the
number of different subcellular particles separated in one
gradient.
[0014] Density gradient separations are important in proteomics
research. High resolution two-dimensional electrophoresis (2DE) is
widely used to produce global maps of the proteins in extracts
prepared by solubilizing whole cells or tissues. By careful control
of the procedures employed, use of staining procedures which are
quantitative, and computerized image analysis and data reduction,
quantitative differences in the abundance of individual proteins of
.+-.15% has been achieved (Anderson, N. Leigh, Nance, Sharron L.,
Tollaksen, Sandra L., Giere, Frederic A., and Anderson, Norman G.,
Quantitative reproducibility of measurements from Coomassie
Blue-stained two-dimensional gels: Analysis of mouse liver protein
patterns and a comparison of BALB/c and C57 strains.
Electrophoresis 6: 592-599, 1985; Anderson, N. Leigh, Hofmann,
Jean-Paul, Gemmell, Anne, and Taylor, John, Global approaches to
quantitative analysis of gene-expression patterns observed by use
of two-dimensional gel electrophoresis. Clin. Chem. 30: 2031-2036,
1984). There is a need for precision subcellular fractionation that
will allow changes in abundance of minor proteins to be accurately
detected and measured in data which sums the abundance of all
proteins found in all of the fractions of one sample.
[0015] This technology allows changes in gene expression, as
reflected in protein abundance, to be studied under a wide range of
conditions, and has led to the development of databases of protein
abundance changes in response to a wide variety of drugs, toxic
agents, disease states. In such studies large sets of data must be
acquired and intercompared. Hence all stages in one pharmaceutical
study, for example, must be standardized for
intercomparability.
[0016] 2DE maps of whole cells or tissues typically contain a
thousand or more protein spots in sufficient abundance to allow
each protein to be analyzed by mass spectrometry and identified and
characterized. However, it is known that a very much larger number
of proteins are actually present in tissue samples analyzed than
are actually observed. The number present varies with cell or
tissue type, and is believed to be up to ten or twenty times the
number detected.
[0017] Different subcellular particles and the soluble fraction of
the cell (the cytosol) contain many location-specific proteins
which constitute only trace fractions of the total cell protein
mass. Hence the total number of proteins resolved from one cell
type or tissue could be greatly increased if the 2DE analysis were
done on cell fractions rather than on whole cell or tissue extracts
as has previously been demonstrated (Anderson, N. L., Giere, F. A.,
et al, Affects of toxic agents at the protein level: Quantitative
measurements of 213 mouse liver proteins following xenobiotic
treatment. Fundamental and Appl. Tox. 8 :39-50, 1987). If a drug
effect study is to be done on cell fractions, however, the
fractionation procedures must be quantitative, in the sense that
the same organelles, or even mixtures of organelles are used in all
analyses to be intercompared. There exists, therefore, an emerging
need for high resolution density gradient separations using
precision gradients in proteomics research. Making precision
gradients reproducibly and in parallel has proven to be difficult,
particularly when the gradients are shallow.
[0018] The protein composition of tissues such as liver varies
diurnally, hence all the tissues from one group of animals are
prepared at the same time of day, and, to be comparable, must be
fractionated in parallel, on the same time schedule, and, if
gradients are to be used, in identical gradients. Further, gradient
fraction recovery must also be done from all gradients in parallel,
under identical conditions. If the initial separations are done
partly or entirely on a sedimentation rate basis, and if the
recovered fractions are to then each be isopycnically banded, as is
done in two-dimensional or s-.rho. fractionation, then these
subsequent steps must also be carried out in parallel. This, in
turn, requires that the gradients be made in parallel.
[0019] Precision gradients are difficult to make in practice, and
it is further difficult to confirm that a set of gradients are all
identical without destroying them for analysis. Existing swinging
bucket rotors generally allow six gradients to be centrifuged
simultaneously. Larger numbers may be centrifuged if the lower
resolution of vertical or near vertical tube rotors is accepted.
Therefore if existing density gradient formers are to be used, a
set of six or more of them operating in parallel will be
required.
[0020] With any gradient maker, small amounts of turbulence or
non-laminar flow typically cause solutions of differing
concentrations to at least partially mix, thereby reducing the
effectiveness and usefulness of the density layers. There is
therefore a need for a method for decelerating fluids flowing into
a tube, and for moving them slowly into position to form distinct
bands.
[0021] One of many uses of density layers and gradients is in the
fields of cell separation, sub-cellular fractionation and analysis,
and density gradient methods are used in molecular biology and in
polymer chemistry. Little attention has been paid to forming sets
of precision-made gradients that are highly reproducible for cell
separation. There is therefore a requirement for precision
gradients adapted to cell separation.
[0022] One high resolution system is disclosed in "Development of
Zonal Centrifuges", by N. G. Anderson, National Cancer Inst.
Monograph 21, 1966) and employs zonal centrifuge rotors. The rotors
are of high capacity, and process one sample at a time. However,
the rotor volumes are too high for many applications. Angle head or
vertical rotor tubes may also be employed (Sheeler, P.,
Centrifugation in Biology and Medicine, Wiley Interscience, N.Y.,
1981, 269pp) using either step or continuous gradients. However
these do not provide the resolution obtained with swinging bucket
rotors.
[0023] There has been no reliable method for reproducibly locating
and recovering organelle zones purely on the basis of the physical
parameters of sedimentation rate and isopycnic banding density.
Mathematical analyses, based on analysis not only of the biological
particles separated, but of the gradients themselves have been
required. These have been tedious and idiosyncratic to the rotors
and conditions employed. The basic problem in preparing density
gradients in tubes is that the liquid volume elements of either
step (layers), or continuous gradients must be introduced into
tubes very slowly or mixing will occur. This problem is only
partially overcome by introducing the gradient into a set of tubes
in an angle-head rotor during rotation.
[0024] Methods for producing one or a few gradients in parallel
have been developed, but fraction recovery is generally done one at
a time. The gradients are rarely identical, and it is difficult to
introduce the sample layer on top of the gradient without mixing.
Hence there is no published data on the quantitative
high-resolution protein analysis of cell fractions of animals
subject to various experimental treatments. If multiple, parallel
identical gradients are to be prepared using gradient engines (for
instance, see "Mechanical device for producing density gradients in
liquids" by N. G. Anderson,. Rev. Sci. Instruments 26: 891-892,
1955) one must have one machine for each tube being filled.
Centrifugal gradient distributing heads have been built (see "A
Method For Rapid Fractionation of Particulate Systems by Gradient
Differential Centrifugation" by J. F. Albright, and N. G. Anderson,
Exptl. Cell Research 15: 271-281, 1958), however the gradients
actually produced tend to be uneven, and a refrigerated centrifuge
is required. There is, therefore, a continuing need for simple
gradient makers that produce identical gradients in parallel in
sufficient number to satisfy current requirements. There is a
further need for a simple, disposable and easily sterilizable
system for making reproducibly sharp step gradients. An additional
need exists for a system or device that can produce very
narrow-step density gradients in which diffusion can rapidly and
reproducibly even out the steps. A further need exists for a system
or device which allows individual gradient steps to be rapidly
pipetted into centrifuge tubes, either manually or robotically, and
in which the introduced fluid does not disturb the underlying
gradient. A still further need exists for a gradient making device
in which the composition of the successive layers, while forming a
stable density series, differ in composition relative to salts,
enzymes, detergents or other reactive materials.
SUMMARY OF THE INVENTION
[0025] One object of the present invention is to provide a rapid,
simple and reproducible method and apparatus for forming a
multiplicity of liquid density gradients in vessels.
[0026] Another object of the present invention is to provide a
rapid, simple and reproducible method and apparatus for forming a
multiplicity of liquid density gradients in vessels for rate-zonal
separations, for isopycnic banding separations, or a combination of
the two.
[0027] Yet another object of the present invention is to provide an
apparatus and method for reproducibly producing a plurality of
liquid density gradients in a plurality of corresponding vessels,
each vessel having a specific predetermined liquid density
gradient.
[0028] An additional object of the present invention is to provide
means for making liquid density gradients in which aliquots of a
liquid density series are rapidly pipetted into the centrifuge
tubes without regard to potential stirring or mixing.
[0029] A further object of the invention is to decelerate the
aliquots ejected from pipettes or automatic pipetters, and to cause
them to flow evenly into position without disturbing the underlying
fluids.
[0030] A further object of the present invention is to provide
means for making the linear or complex gradients by making them
initially as step gradients having very small density differences
per step.
[0031] A further object of the present invention is to produce step
gradients in which the steps are so small that diffusion rapidly
evens out the gradient.
[0032] A still further object of the present invention is to make
the gradient making components disposable and easily
sterilizable.
[0033] It is a further object of the present invention to make
possible construction of sets of identical gradients in a short
period of time.
[0034] It is an additional object of the present invention to make
possible addition of the sample layer on top of the gradient at any
time after the gradient is formed.
[0035] In accordance with one aspect of the present invention,
there is a method for producing liquid density gradients in a
vessel using a float within the vessel includes the steps of:
[0036] inserting the float in the vessel;
[0037] introducing a first liquid into the vessel;
[0038] introducing a second liquid into the vessel such that the
second liquid contacts at least one surface of the float upon
entering the vessel, contact between surfaces of the float and the
second liquid allowing the second liquid to form a layer above the
first liquid thereby forming separate layers of liquid; and
[0039] repeating the second introducing step with successive
introducing steps with a third, fourth and so on liquid.
[0040] The float used in the above method slows the velocity of
fluid such that flow of liquid is laminar thereby limiting mixing
of the two liquids.
[0041] In accordance with another aspect of the present invention,
an apparatus for producing liquid density gradients includes a
vessel and a float positionable in the vessel. The float is formed
with at least one surface that is shaped to inhibit acceleration of
fluid introduced into the vessel thereby restricting turbulent flow
of the fluid.
[0042] An outer peripheral surface of the float and the inner
surface of the vessel are sized such that in response to fluid
being introduced into the vessel above the float, the fluid
undergoes capillary action moving downward beneath the float in the
vessel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Further advantages of the disclosed invention will become
apparent from a reading of the following description when read in
conjunction with the accompanying drawings where like reference
numerals are used to identify like parts, in which:
[0044] FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G are side views of a
vessel and a float for producing a density gradient in the vessel
in accordance with the present invention;
[0045] FIGS. 2A, 2B and 2C show details of the design and operation
of float;
[0046] FIGS. 3A, 3B and 3C are side views showing alternate
embodiments of the float;
[0047] FIGS. 4A and 4B are a side view showing yet another
embodiment of the float; and
[0048] FIG. 5 is a side view showing still another embodiment of
the float.
DETAILED DESCRIPTION OF THE INVENTION
[0049] A first embodiment of the present invention is illustrated
in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. In accordance with the
present invention, a float 1 is used to form a step gradient within
a vessel 2, as depicted in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G.
However, it should be understood that the float 1 may also be used
to create a continuous gradient (not shown) where a density
gradient is introduced gradually, and continuously changes along
the height of the vessel. In FIG. 1A the float 1 in the vessel 2
rises and floats on top of a liquid 3 introduced from a source 4.
The diameter of the float 1 in the first embodiment is preferably
slightly smaller than the inside diameter of the vessel 2.
[0050] The introduced liquid 3 contacts and then flows around the
float 1, passes down between the outer surface of the float 1 and
the inner surface of the vessel 2 and, as shown in FIG. 1A,
produces a first zone 5. Typically, the zone 5 has the highest
density of all the layers or zones, as is described in greater
detail below. As shown in FIG. 1B, a second liquid 6 is introduced
in a similar manner to produce zone 7. As shown in FIGS. 1C, 1D,
and 1E, the procedure is repeated with succeeding less dense
liquids 8, 10, and 12, to produce zones 9, 11, and 13.
[0051] A sample 14 to be analyzed within the vessel 2 is introduced
last from a pipette 15 to produce zone 16 as shown in FIGS. 1F and
1G. Finally as shown in FIG. 1G, the float 1 is removed by grasping
a projecting pin 17 and lifting. The vessel 2 depicted in FIG. 1G
with sample and gradient may then be subjected to treatment by, for
instance, insertion into a swinging bucket rotor of a centrifuge
device. When a continuous gradient is required, the step gradient
is prepared beforehand, and diffusion for a determined period of
time used to convert the step gradient into a continuous one.
[0052] FIGS. 2A, 2B and 2C illustrate details of the float 1 and
basic principles of operation of the float within the vessel 2. The
float 1 and vessel 2 depicted in FIG. 2A is shown enlarged in FIG.
2B. The float 1 has an outer peripheral surface 20 that is shaped
to conform to an inner surface of the vessel 2. For instance, the
vessel 2 shown in the drawings is a tube having a circular
cross-section when viewed from top or bottom. The float 1 has a
corresponding circular shape with the outer peripheral surface 20
having a diameter that is smaller than the inner diameter of the
surface of the vessel 2. Therefore, a gap G having a predetermined
width is defined between the outer peripheral surface 20 of the
float 1 and the inner surface of the vessel 2. The gap G may vary
in size depending upon the solutions to be introduced into the
vessel 2 and the relative sizes of the float 1 and vessel 2.
[0053] In the depicted embodiment, the gap G is relatively small
such that surface tension of the solution produces a capillary
action within the gap G to maintain liquid in the gap and to
prevent air bubbles in the gap. In many applications of the present
invention the viscosity of fluid in the gap is sufficient to
eliminate the possibility of turbulent fluid flow within the vessel
2 as the solution exits the gap and moves around the float 1.
Therefore, mixing of layers of solution under the gap is almost
non-existent and very sharp boundaries are produced between the
zones, even with very small density increments.
[0054] It should be understood that diminished rate of fluid flow
is a desirable result of the present invention depicted in FIGS.
1A-1G and FIGS. 2A-2C. The actual size of the gap G may be varied
according to the viscosity of the liquids used and the size of the
vessel 2. However, although capillary action and restricted rate of
fluid flow are important to the present invention, it is possible
to use the float 1 of the present invention without capillary
action. For instance, the shape of the surfaces of the float 1 may
be formed to discourage any increases in velocity of fluid moving
over the surfaces of the float 1 to avoid turbulent flow of the
fluids entering the vessel 2. The shape and surface contours of the
float 1 are such that the flow of solution around the float 1 as
the solution moves downward into the vessel 2 is minimal.
Specifically, a upper surface 22 of the float 1 is tapered having a
conical shape such that as fluid contacts the upper surface 22
viscous flow slows fluid motion as the fluid approaches an edge 23
of the float 1.
[0055] It should be understood that the upper surface 22 may have a
more rounded shape when viewed from the side and need not be
conical in shape so long as sufficient surface area is provided to
allow the adhesive forces of the fluid to make contact with the
upper surface 22 to slow movement of the fluid.
[0056] It should also be understood that the vessel 2 and float 1
may have any of a variety of shapes when viewed in cross-section.
The depicted vessel 2 is a tube having a circular cross-section.
The vessel 2 may also have a square or triangular cross-sectional
shape and the float 1 a corresponding square or triangular
cross-sectional shape.
[0057] As shown in FIG. 2B, when a droplet 21 of solution is
dropped from above the float 1, the droplet 21 is distributed
circumferentially on upper tapered surface 22 and moves toward the
edge 23, where the solution flows evenly into the gap G, and
thereafter slowly moves on to the upper surface of the underlying
layer of liquid. Velocity or speed of flow of the solution is also
further decelerated as it flows around lower taper 25. Capillary
forces and solution viscosity are sufficient to keep the velocity
of the solution in the gap G to a minimum and further, regardless
of the density of the liquids used, the gap G typically remains
filled with solution due to the capillary action.
[0058] The float 1 is also formed with an upper integral pin 17
that allows the float 1 to be inserted and removed easily from the
vessel 2. The density of the float 1 itself may be dictated by the
choice of construction material or, as shown in FIG. 2C, an
alternate embodiment of a float 1a may be formed with a cavity 26
sealed by plug 27 to adjustably control the density of the float 1.
The floats 1 and 1a are preferentially constructed of polypropylene
which has a density of approximately 0.95 g/cc, and the pin 17,
being a small fraction of the mass of the float, may be either
integrally molded into the float and of the same material, or may
be another material such as polycarbonate or other plastic, and be
inserted in a hole in the float as shown in FIGS. 4A and 4B.
Further, the density of the float may be adjusted by inserting pins
17 having a variety of weights. For instance, a plurality of pins
17 may be produced, each pin 17 having a different mass for
selectively adjusting the overall weight of the float.
[0059] The shape of the various surfaces of the float 1 is not
limited to the depictions in FIGS. 1A-1G and FIGS. 2A-2C. FIGS. 3A,
3B and 3C illustrate alternative float designs. In FIG. 3A a float
28 has upper edges 29 and lower edges 30 rounded to further assist
in slowly accelerating flow at the upper edge, and decelerating
flow at the lower edge as liquid flows over the underlying liquid.
In FIG. 3B float 31 has different upper edge 32 and lower edge 33,
with the upper edge 32 sharp to help prevent air bubbles between
the float and tube 2, and the lower edge 33 well rounded, while in
FIG. 3C float 34 has a tip 35 extended to further control flow
around the float. The shape of the lower surface of the float 34
and the tip 35 assist in keeping the interface 36 between two steps
in the gradient sharp.
[0060] The inventors have tested and designed floats for Beckman
Ultraclear tubes for the Beckman SW41 Ti rotor and for
polycarbonate tubes for the Beckman SW28 rotor. For the SW 41
tubes, the floats were constructed of solid polypropylene, 13.1 mm
in diameter with top and bottom tapers of 15 degrees, and were 6.35
mm high measured at the edge. Wall clearance was 0.25 mm (gap G).
For the SW 28 rotor tubes, long and short versions of the floats
were constructed which were 10.5 mm and 6 mm high at the edge, had
clearances of 1 and 0.6 mm, with 15 degree tapers at the top and
bottom. Holes through the float had 1.6 mm internal diameters, and
the pins were made of 0.9 mm outside diameter polycarbonate
monofilament. After the pins were inserted, one end was melted in a
reducing flame to produce a ball at the tip, while the other end
was heated to produce a small enlargement, which, when put into the
float, sealed the pin in place.
[0061] All radial clearances kept the gap G between the float and
the centrifuge tube wall (vessel 2) full of liquid at all densities
used. Occasionally when floats were dropped into dry tubes, they
became stuck at the bottom, hence the "round" at the bottom of the
centrifuge tube is preferably filled with a "cushion", i.e.,
densest gradient solution used, initially.
[0062] Experimentally it was found that if the first 4-5 drops
(circa 0.1 ml) of the solution being added were introduced slowly
over a period of 5-10 seconds, extraordinarily sharp interfaces
were produced below the float. The remainder of the gradient step
could then be introduced more rapidly. Sharp interfaces were
produced with the density difference between two steps being as
little as 0.0017 g/ml.
[0063] The use of floats allows gradients to be formed as a series
of short well defined zones that may be arranged to be linear,
sigmoidal, or of other gradient shape. If required, the gradients
can then be evened by diffusion. The float/vessel arrangement
allows the production of gradients that are more reproducible than
those produced by conventional gradient makers, and allows many
gradients to be made in parallel without requiring a multiplicity
of gradient makers.
[0064] However, it should also be understood that by using the
float of the present invention, it is possible to quickly pour an
amount of a fluid directly onto the top of the float and the fluid
will gradually seep down around the float to create a layer fluid
without significantly disturbing the layer or layers of fluid
already beneath the float. Without the float, pouring of fluid into
the vessel with previously introduced fluid layers in the vessel
would guarantee mixing of the layers thereby making gradient layer
formation impossible. Therefore, one important result possible by
using any of the above described embodiments of the present
invention is that a density gradient can be produced quickly and
reproducibly without concern of the rate of flow of any one liquid
onto the upper surface of the float.
[0065] In yet another embodiment of the present invention depicted
in FIG. 5, a float 50 is positioned in a vessel 52 with the vessel
52 having an inner diameter that is significantly greater than the
outer diameter of the float 50. The float 50 is similar to the
float described above in FIGS. 1A-1G, but has a tube 55 attached to
an upper surface of the float 50. The tube 55 is hollow and
includes several apertures 58 for allowing the flow of fluid from
within the tube 55 to an upper surface of the float 50. As fluid is
introduced from the tube 55 via the apertures 58, the fluid
contacts the upper surface of the float 50 and flows along the
upper surface due to adhesion thereby slowly entering the vessel.
Adhesion between the surfaces of the float 50 and the fluid slows
velocity of the fluid such that the fluid forms a well defined
layer above previously introduced layers.
[0066] The upper surface 51 of the float 50 is preferably formed
with only a slight incline to further inhibit acceleration of the
fluid. The tube 55 attached to the fluid may also be used to raise
and lower the float 50 with respect to the vessel 52. Specifically,
a mechanical arm may be attached to the tube 55 to remotely control
movement of the float in and out of the vessel 51. It should be
understood that the tube 55 is flexible to allow movement of the
float 50 upward as the vessel 52 is filled with fluid.
[0067] A fluid flow controller (not shown) is preferably used with
the embodiment of the float 50 to control the amount of fluid
introduced for each desired layer.
[0068] It should be understood that gradients may be used in many
applications outside of the field of molecular biology. For
instance, a vessel having a plurality of layers solution, each
layer having a different density due to a specific concentration of
solute in each layer, may be used determine the density of an
unknown material. A sample of material of unknown density dropped
into the vessel will settle in the layer having a like density
thereby providing a means for determining density of the unknown
material. For example, different classes of plastics have different
densities. Small pieces of plastic may easily be tested by dropping
one small sample into a vessel having a plurality of solutions,
each solution having a predetermined density such that the
plurality of layers define a stepped density gradient. The plastic
particle will drop to a layer having the same density and will
float above those layers having a heavier density. Similarly,
identification of a gemstone based on density can be conducted.
[0069] The materials and methods of the instant invention can be
used in the separation of cellular elements from samples of whole
blood, blood products or diluted blood. For example, white blood
cells can be obtained from blood by density gradient
centrifugation. Suitable materials to effect separation of the
cellular elements and particularly the nucleated cellular elements
from blood include media that comprise colloidal silica, silica
gel, sugars, such as sucrose, ficoll, and particular products such
as Ficoll-Hypaque, Isopaque, LymphoPrep and Percoll. See, for
example, Parish et al., Eur. J. Imm. (1974) 4:808.
[0070] Generally single step gradients are produced by gently
layering the blood cell suspension onto a high density medium. The
preparation then is centrifuged at low speed to effect separation
of the cells.
[0071] Alternatively, the blood cell suspension can be layered onto
a linear gradient, for example, of bovine serum albumin prior to
centrifugation. The blood cell suspension can be layered onto a
discontinuous gradient, for example, of bovine serum albumin. The
densities of the layers can be configured so that the various
elements band at the interfaces of the layers.
[0072] To ensure that discrete, sharp layers and hence tight
banding of cells occurs, it is beneficial to ensure a sharp
interface between the cell suspension, for example, blood, and the
separation medium. That goal can be achieved with use of an
apparatus of interest. A suitably sized float of interest is used.
The float of interest rests atop the separation medium. The float
of interest allows passage of the, for example, blood along the
lateral sides thereof and along the inner surface of the centrifuge
tube containing the medium, float and cell suspension with minimal
turbulence to ensure formation of a discrete linear interface of
cell suspension and separation medium.
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[0135] All references cited herein are herein incorporated by
reference in entirety.
[0136] Although the present invention has been described with
reference to the preferred embodiments, the invention is not
limited to the details thereof. Various substitutions and
modifications will occur to those of ordinary skill in the art and
all such substitutions and modifications are intended to fall
within the scope of the invention as defined in the appended
claims.
* * * * *