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FIELD OF THE INVENTION
This invention relates to apparatus useful for immobilizing biological
cells on a surface and for selectively releasing or imparting
predetermined motion to biological cells and is particularly useful in
conjunction with the generalized cytometry instrument of the commonly
assigned co-pending application of Louis A. Kamentsky entitled
"Generalized Cytometry Instrument And Methods Of Use", filed concurrently
herewith and having Ser. No. 577,448.
BACKGROUND OF THE INVENTION
In U.S. application Ser. No. 577,448 of Louis A. Kamentsky describing a
generalized cytometry instrument, a necessary aspect involves the
immobilization of the cells to be examined onto a surface. Kamentsky
describes the use of immobilizing media such as agarose for suspending the
cells in a fixed position and for providing viable nutrients or reagents
to same. Alternately, Kamentsky discloses the use of immobilized
antibodies for selectively or nonspecifically securing desired cells to
the surface for subsequent illumination, detection, and analysis.
It is an object of the present invention to provide another means whereby
biological cells may be immobilized to a surface.
It is a yet further object of the present invention that apparatus and
methods be provided for immobilizing biological cells in a manner whereby
they may be selectively released for purification procedures and the like
in response to detected cellular characteristics.
It is a yet further object of the present invention to provide methods and
apparatus which, in conjunction with data results obtained by the
generalized cytometry instrument, permit the specified translocation of
cells thereby effecting purification and segregation of those cells either
desired or undesired from a heterogeneous population.
SUMMARY OF THE INVENTION
In accordance with the principles and objects of the present invention,
there are provided apparatus and methods for immobilizing biological cells
based upon their dielectric properties in an inhomogeneous or non uniform
electric field. Cells, when placed in an inhomogeneous electric field
generated by, for instance, an alternating current source, exhibit a net
force in the direction of the field's source hereinafter referred to as
grid points. By arranging an array of grid points having the numerical
density desired to attract and immobilize the number of cells required for
analysis in a desired pattern, and by connecting same to an inhomogeneous
field generating source, one may vastly simplify cell locating operations
such as those performed by Kamentsky's generalized cytometry instrument
since the cells' position will now be associated with the grid point
locations.
By individually connecting the grid points to controlling means, one may
selectively energize grid points or other conducting contact areas and if
the inter-grid point distances are appropriately selected and the proper
fields applied in a coordinated timing sequence, one may ideally effect a
predetermined translocation of the cells. This will advantageously permit
the removal of undesired cells by translocating them to "sinks" for
disposal and the purification of desired cells by their movement to other
predetermined collection locations.
BRIEF DESCRIPTION OF THE DRAWINGS
Further understanding of the invention may be had by reference to the
drawings wherein:
FIG. 1 shows a stylized view of the inhomogeneous fields resulting from
energized grid points;
FIG. 2 shows cellular interactions when placed within an inhomogeneous
field; and
FIG. 3 graphically depicts an embodiment of the present invention.
FIGS. 4 and 4a depict an embodiment of the present invention as used in
Experiment 1.
DETAILED DESCRIPTION OF THE DRAWINGS AND BEST MODE
With reference to FIG. 1, there is shown a series of grid points generating
an inhomogeneous field graphically depicted by the curving field lines 10
from grid points 11. The inhomogeneous or non uniform field may be
advantageously generated by connecting grid points 11 via connections 12
to an alternating current source oscillating in the kilohertz range.
It is well-known that dielectric particles, such as biological cells 25,
see FIG. 2, experience a force of attraction in a nonuniform electric
field. This force, depicted as F.sub.net in FIG. 2, produces motion of the
cell in the direction of the highest field strength. As may be readily
seen by examination of FIG. 2, the field strength at point 27 is larger
than that experienced by the cell at point 26 and accordingly the cellular
surface charges are greater at 27 than at 26. Thus, in accordance with the
attraction between unlike charges, cell 25 experiences a force F.sub.net
in a direction of grid point 21.
With appropriate manipulation of the cellular concentration in solution as
well as the density of grid points, each grid point will preferably
attract on the average one or less cells. At higher cell concentrations,
the cells will disadvantageously tend to form chains extending from the
location of the grid point toward the region of lowest electric field
strength and unless this effect is specifically desired, it is
advantageously avoided. Release of the cells from the grid points may be
readily accomplished by merely disconnecting the grid point from the
current source thereby turning off the electric field and eliminating the
grid point's attractive influence upon the cell.
It should be noted that, in accordance with the types of cells to be
immobilized, the suspending solution must be appropriately adjusted to
permit the generation of the inhomogeneous electric field. It will ideally
also be chosen to provide an environment suitable for continued or
maintaining cellular viability for a time at least sufficient to conduct
the desired testing or culturing of the cells, particularly if it is to be
employed with the class of generalized cytometry instruments such as those
described by Kamentsky.
As may be seen in FIG. 3, the grid points are preferably embedded in an
insulating substrate 30 for careful maintenance of inter-grid point
distance d (see also FIG. 1) as well as for providing a degree of physical
integrity for repetitive use. Insulating substrate 30 may, in fact, be the
walls of a container such as those found in the so-called microtiter type
tray, or the bottom thereof, or in the case of the generalized cytometry
instrument, any surface suitable for the illumination and detection of
cells with that class of instruments.
The grid points 31 are ideally connected via connections 32 in any of a
variety of ways, some examples of which are shown in FIG. 3, to a position
control processor 33. The position control processor controls the grid
point connections to alternating current source 34 in a manner appropriate
for analysis.
For example, the position control processor may be programmed to provide
cellular inter-grid point movement by sequential connection of grid points
to the inhomogeneous field generating source whereby cells may follow
preprogrammed routes. Thus, the cells may, for example, pass through
specified environments containing test reagents or environments suitable
for various testing methods. Additionally, the ability to selectively move
the cells on a grid pattern, would enable an investigator to perform cell
sorting based on cell kinetic response or other optically sensed cell
characteristics. Since with the generalized cytometry instrument of
Kamentsky, the location of the cells and the history of each cell is
stored in the central processing unit or computer, one can de-energize the
grid points or conducting contact areas having unwanted cells associated
therewith, wash the system and perform a second set of analytical
procedures on the remaining selected cell populations thereby effecting
further subcell populations definitions. Similarly, undesired cells may be
removed by translocating the cells past a sequentially energized series of
grid points to a disposal area. Sorting based on the remaining cells
utilizing such procedures may be performed on a repeated basis as
necessary to achieve the desired level of purification and/or selection.
Upon achieving the final desired subset separation, the cells could be
transported to adjacent grid systems for collection and for culturing.
Indeed, further employment of the immobilizing surface of the present
appartus is contemplated as a means whereby cells such as hybridomas and
the like may be continuously cultured and their exuded products
(antibodies in the case of hydridomas) facilely collected.
In conjunction with the class of generalized cytometry instruments
described by Kamentsky, the present invention would permit automated
cellular analysis and sorting to be continuously carried out for prolonged
periods of time to obtain extremely sensitive sorting of particularly
desired but rare cells in large heterogeneous populations. Accordingly,
such a system would be vastly superior to the systems employing flow
cytometric type cell sorters as those systems cannot be continuously
operated for more than a few hours thereby limiting the utility of such
systems as reagent preparative or investigational devices.
The invention can be used in still further applications and in particular,
by appropriate selection of inter-grid point distance d, permits an
investigator to bring cells into physical contact for studies of
cell-to-cell communication. The grid points further permit the
investigator to electronically pulse particular cells thereby stimulating
pores to be open in the cell's membrane. The appearance of pores
facilitates the transport of intracellular material betwen adjacent cells.
If this process is carried to an extreme, actual cell fusion may result.
As may be readily apparent, the application of electronic pulses and the
resultant formation of membrance pores will readily permit the
introduction of biochemicals through the cell membrane. This operation of
forced increased permeability may be programmed to be carried out at any
stage of the cellular analysis or cell sorting process. Such a process
would, for instance, permit the analysis of a cell for phenotypic
determinants such as surface receptors or other markers, and then
subsequently allow genotypic analysis through the introduction of nucleic
acid probes such as those available from Enzo-Biochem of New York, N.Y. It
should be noted that these probes need not be introduced together but may
be introduced separately thereby allowing analysis to occur between the
introduction of each probe. With the kinetic analysis properties of the
generalized cytometry instrument of Kamentsky, it may be possible to
actually track the time course genetic expression through the manufacture
of messenger RNA and the subsequent appearance of gene products for each
cell in an ensemble on the grid.
The shape and size of the grid containing the grid points may be adjusted
to a virtually infinite number of embodiments. One useful embodiment, for
example, would be the generation of a series of stripes of electronic grid
points e.g. elongated contact areas arranged in a pattern, for use in that
class of instruments employing synchronous detection. Alternatively, the
grid points may be replaced with a series of finite repeating conducting
bars, especially useful for the synchronous detection instruments. The
cells would be introduced in suspension and then electrostatically
immobilized on the bars.
Synchronous detectors derive their advantages from the repetitive detection
of a pattern of cells via a lock-in amplifier to permit homogeneous
testing and the extraction of a signal from a background or noise signal
which may be much greater. This is accomplished by placing a rotating
reticle in the image plane of the microscope with a pattern matching the
image of the electrostatic bar pattern. Assuming the cells have been
reacted with a probe (such as an antibody or nucleic acid probe)
containing an optical label, rotating the reticle will provide an optical
signal periodically transmitted through the reticle to a photomultiplier
tube. The photomultiplier output would be synchronously detected by
incorporating a reference signal from the rotating reticle. Thus,
synchronous detection eliminates the need to wash free label from the
ambient solution by suppressing any spatially uniform background optical
signals. Further extensions of this embodiment would include a number of
separate bar patterns on the microscope stage or other surface, each
containing a separate cell sample undergoing analysis. A stage translator
and method for detecting the optical register of the bar pattern in the
reticle could be used to automatically interrogate each separate bar
pattern over a predetermined time course for kinetic studies. These types
of arrangements would permit the analysis of the average properties of
cells rather than analyzing the cells on an individual basis.
If the patient sample is cellular in nature, it may be advantagous to
transport the cells to each bar pattern location by utilizing the
electrostatic grid technique. In this manner, samples can be automatically
fed to the system and then removed following analysis. As with previously
described preferred embodiments, traffic patterns in analysis results
would be ideally computer controlled, such as by the position control
processor.
EXPERIMENT 1--CELL MOTION--METHOD 1
With reference to FIG. 4, 5 wires were glued to a microscope slide in
parallel with all ends (the tips) aligned. Wire 6 was glued to the slide
perpendicularly to the 5 wires and at one end of the parallel wires.
Wires 1-5 were left insulated except for the tips which were 200.mu. from
wire 6. Wire 6 was entirely uninsulated. The ends of wires 1-5 were spaced
200.mu. apart. Wire 6 was grounded and wires 1-5 were left electrically
floating. 2-1-14 (mouse myeloma) cells were washed twice in 300 mM
Mannitol (Baker #2554-1) pH 7.5, conductivity .about.8-10
.mu..upsilon./cm, and resuspended to a concentration of 2.times.10.sup.6
cells/ml. Cells were kept on ice until needed.
A drop (.about.100.lambda.) of cell solution was placed on the uninsulated
wire tips. In turn, each of the wires 1-5 was connected to a Tektronix
signal generator (TM5006) [20 V peak-to-peak, 2 MHz sine wave] while the
remaining wires were left unconnected.
The cells were seen to dielectrophorese to whichever wire was connected to
the signal generator--See FIG. 4a which shows two circumstances of
cellular alignment.
When wire #1 was disconnected, and wire #2 connected, the same
configuration of aligned cells was seen at wire #2. After removal of
signal to wire #1 the cells began to disperse gradually but slowly.
Individual movement of cells is possible with this configuration when the
cells are present at extremely low concentrations; on the order of
1.times.10.sup.3 cells/ml. In some cases, the wires themselves presented a
mechanical barrier to cell motion if the cells were initially positioned
such that a "floating" wire was between them and the "active" wire. This
can, of course, be obviated by placing the wire or contact within the
surface.
EXPERIMENT 2--CELL MOTION--METHOD 2
The problem of wires presenting a physical impedance to cell movement was
eliminated in a chamber constructed using a microscope slide upon which 8
copper conducting tape wire islands were applied in radial fashion, the
outer most ends uniformly separated and forming the outline of a circle. 1
mil wire bonding aluminum wire was used to connect the islands and then
covered with a ring of epoxy leaving the wire ends exposed for subsequent
electrical connection as well as leaving exposed the wire islands crossing
in the center of the epoxy ring.
Acid was used to etch away the wire islands in the center leaving a
circular wall of epoxy having exposed, opposing point contacts. Distance
across the center well between contacts was approximately 200.mu..
2-1-14 (mouse myeloma) cells were washed twice in 200 mM Mannitol (Baler
#2554-1) pH 7.5, conductivity 8-10 .mu..upsilon./cm. Cells were
resuspended to a concentration of 2.times.10.sup.6 cells/ml in 300 mM
Mannitol, and kept on ice until used.
A drop of cell solution was used to fill the chamber. In turn, each pair of
opposing wire ends was connected to a "Viking Challenger" radio
transmitter employed as a signal generator to create a 30 V peak-to-peak 2
MHz sinusoidal field across the chamber.
Cells migrated to the "active" point contacts and then to other pairs of
point contacts as they were in turn connected to the signal generator. The
cells aligned in a string of pearls fashion between the active point
contacts. In this manner cells could be moved around the entire interior
of the chamber. Directed movement of single cells can similarly be
accomplished at cell concentrations on the order of <1.times.10.sup.3
cells/ml.
EXPERIMENT 3--MAINTAINING CELL LOCATION
In many applications actual cell testing or interrogation cannot be
accomplished in the non-ionic or low conductivity solutions otherwise
necessary to create the electrical cell locating fields. (It will readily
be understood that although electric fields may be generated in high ionic
solutions, such fields are accompanied by disadvantageous joule heating
effects detrimental to continued cellular viability). Accordingly, the
following demonstrates how cells may be located using the principles of
the present invention and then, how their location can be maintained in
the absence of an electric field using agarose. Thereafter, the agarose,
on a suitable heating stage, may be melted at low temperature, either
entirely or only at a specified location to allow repositioning of the
cells via a subsequently applied electric field. 653 (mouse myeloma) cells
were washed twice in a solution of 300 mM Mannitol (Baker Reagent Cat.
#2554-1) pH 7.5 having a conductivity of 8-10 .mu..upsilon./cm. The cells
were then resuspended in 300 mM Mannitol to a concentration of
2.times.10.sup.6 cells/ml. The final cell suspension was essentially ion
free and therefore not subject to deleterious joule heating effects when
placed in an alternating electric field of sufficiently high frequency.
The cells were kept on ice until used.
Agarose type VII (Sigma #A-4018) was dissolved in 300 mM Mannitol to a 1%
solution. The solution was then microwaved for 30 seconds to completely
dissolve all the agarose and then kept warm in a boiling water bath until
needed.
The heating stage was warmed to 30.degree. C. and a small volume (about
100.lambda.) of cell solution was placed between parallel electrodes glued
to the bottom of a 60 mm Petri dish. Distance between the electrode wires
was 200.mu..
A 10 volt (peak-to-peak) 2 MHz (sine) field was then applied across the
electrodes with a Tektronix TM5006 signal generator. This field caused
cells to align themselves along field lines (perpendicular to the
electrode wires) in pearl chain type arrangements such as those shown in
FIG. 4a. Cells were in mutual contact.
The 1% agarose solution was then cooled to <37.degree. C. and layered over
the aligned cells. Addition of the agarose resulted in rotation of about
half of the cells on an axis normal to the field lines. Rotating cells and
adjacent cells were no longer in cellular contact. This effect could be
totally eliminated by decreasing the field frequency to 1 MHz. By lowering
the temperature to 27.5.degree. C., the agarose gels immobilized the cells
in the pearl chain arrangements, thereby permitting removal of the
electric field without disruption of the cellular arrangement.
If the cells were permitted to rotate thereby creating intracellular
spaces, the spaces could also be maintained upon solidification of the
agarose. Reheating the solution to 31.degree. C. melted the agarose
sufficiently to allow cell repositioning via an applied electric field. By
lowering the temperature to 27.5.degree. C., the agarose was again
solidified and cell position again maintained.
Standard commercial agarose can be remelted at 31.degree. C. at least twice
without requiring cell-destructive heat levels. Thereafter, it is
theorized, the agarose becomes increasingly cross-linked requiring greater
heat before melting is accomplished. However, this cross-linking effect
can be reduced and the agarose "remelting-life" extended by limiting the
formation of cross-linkages in manners well known by those skilled in the
art.
The maintenance of intracellular space may be advantageous in some
applications requiring optical interrogation of the cells. For those
applications not requiring the intracellular space, it can be readily
eliminated by application of lower field frequencies as aforesaid.
It will be readily apparent to those skilled in the art that numerous
alternatives are possible without departing from either the spirit or the
scope of the present invention.
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