CA2845713A1

CA2845713A1 - Gradient array dielectrophoresis separation (grads) with concomitant light therapy

Info

Publication number: CA2845713A1
Application number: CA2845713A
Authority: CA
Inventors: David Martin Morrow; Allan Yang Wu
Original assignee: COSGENIX LLC
Current assignee: COSGENIX LLC
Priority date: 2011-08-19
Filing date: 2012-08-17
Publication date: 2013-02-28
Also published as: JP2014531199A; IL230985A0; WO2013028573A4; WO2013028573A1

Abstract

A method and device in which a heterogeneous population of cells or particles is introduced into a series of chambers with successively increasing dielectrophoresis fields produced by planar microelectrode arrays and thus separated by charge and size. A clear or translucent third electrode overlying the planar array defines the ceiling of the flow chamber and allows the simultaneous introduction of light therapy to cells while they are being separated. Flanking planar electrodes to the capture electrodes may be utilized for repulsive dielectrophoresis purposes in order to confine and keep separate entrapped cells during the final collection and elution phase. The entire process may be monitored electronically by impedance testing or visually by microscopy in-situ and real-time without disrupting the separation process.

Description

Gradient Array Dielectrophoresis Separation (GrADS) with Concomitant Light Therapy Technical Field The present invention relates the field of dielectrophoresis and more particularly to a method and device using dielectrophoresis to separate fractions of particles/cells from a heterogeneous population of particles/cells and simultaneously allow light or electrical therapy to the cells. In one example of the application, the cells may be derived from I iposuctioned adipose tissue.
Background Art Dielectrophoresis (DEP) has been utilized for charged particle separation since 1950 (H. A. Pohl, "The Motion and Precipitation of Suspensoids in Divergent Electric Fields", J. Appl. Phys. 22(7), 869-871 (1951)). The first use of this for mammalian cells was reported by Ronald Pethig in 1995 in US Patent No.
5,814,200.
One problem with two dimensional (2-D) DEP in that cell clumping can occur within the electrodes which can be further accelerated with a "pearl of chain"

effect and buildup of charge. (Robert Kretschmer and Wolfgang Fritzsche Langmuir, 2004, 20 (26), pp 11797-11801) An additional problem with 2-D DEP is that parasitic cell traps can occur.
(Mario Urdaneta and Elisabeth Smela Lab Chip, 2008, 8, 550-556) Use of three-dimensional (3-D) DEP has been reported in US Patent No.
1,617,978, US Patent Application Publication No. 2006/0260944 (Serial No.11/419,144) and US Patent No. 7,686,934 (Serial No.11/638,093). However, in these documents it was noted that the electrodes were difficult and complex to manufacture.
Elution by frequency modulation is one method of separating cell fractions from a heterogeneous population. See "Enrichment of putative stem cells from adipose tissue using dielectrophoresis field-flow fractionation", Jody Vykoukal, Daynene M. Vykoukal, Susanne Freyberg, Eckhard U. Alt, and Peter R. C. Gascoynea, Lab Chip. 2008 August; 8(8): 1386-1393 (Published online 2008 May 28. doi:

10.1039/b717043b). This is done by starting with a high "entrapping" frequency of 200 kHz and slowly stepping down the frequency. Each unique fraction is collected as the current is stepped down. However, in this method it is difficult to adjust the timing so as to collect the different elution fractions.
Currently, there is no device that provides low cost 3-D DEP with simultaneous light/electrical therapy to cells while in the chamber, and at the same time avoids step down voltage fraction collection.
This invention is an improvement of the inventor's prior US Patent Applications Nos. 61/545,015, 61/136,932 and 12/578,549; and PCT Patent Application No.
PCT/US09/6071. The non-provisional and PCT applications were published as US Patent Application Publication No. 2010/0112084 and WIPO Publication No.
WO/2010/045389. The entire specifications, claims and drawings of these applications and publications are hereby incorporated into this application by reference.
The improvement also comprises modification of the prior invention apparatus to detect differing electrical "signatures" of each different phase (i.e. oil vs.
fat vs.
aqueous solution) as they pass through a channel and actuate a diverting port at the end of the channel to collect and separate the different phases of lipoaspirate.
The electrical signatures may be measured by means of: current, current signal shift, conductivity, resistance, impedance, phase shift, magnetic shift with either direct or alternating current.
The following equations help illustrate some of the physical principles behind the method and device.
Dieletric Force:
For a field-aligned prolate ellipsoid of radius r and length / in which r < I, with complex dielectric constant in a medium with complex dielectric constant the time-dependent dielectrophoresis force is given by:

r - - 2 FDEF = ¨enz ______________________________________ El =
The complex dielectric constant is, where c is the dielectric constant, a is the_electrical conductivity, w is the field frequency, and j is the imaginary number.
This equation is accurate for highly elongated ellipsoids when the electric field gradients are not very large (e.g., close to electrode edges). The equation only takes into account the dipole formed and not higher order polarization. When the electric field gradients are large, higher order terms become relevant, and result in higher forces. To be precise, the time-dependent equation only applies to lossless particles, because loss creates a lag between the field and the induced dipole. When averaged, the effect cancels out and the equation holds true for lossy particles as well. An equivalent time-averaged equation can be easily obtained by replacing E
with the root mean square of E (Erms), or, for sinusoidal voltages by dividing the right hand side by 2.
For a homogeneous sphere of radius r and complex permittivity in a medium with complex permittivity, the (time-averaged) DEP force is:
=
(FD324 '5= 117- s ¨1= Epim t2e The factor in curly brackets is known as the complex Clausius-Mossotti function and contains all the frequency dependence of the DEP force.
Particle Levitation:
Particles are levitated above the planar electrode array by negative dielectrophoresis. The levitating negative DEP force, FDEPz (which has been shown to fall exponentially with height above the electrode array) and hydrodynamic lift force are balanced against a sedimentation force, Fgrav, to establish particle equilibrium position, heq within the parabolic flow profile.

Under moderate flow rate, at approximately 1cc/hour, the equilibrium height, heq, for a given particle type is expressed by:
f 2 (Pp ¨ Pin kg 341P Re (km) 1J2 The prior invention apparatus may be modified to detect differing electrical "signatures"
of each different phase (i.e. oil vs. fat vs. aqueous solution) as they pass through a channel and actuate a diverting port at the end of the channel to collect and separate the different phases of lipoaspirate. The electrical signatures may be measured by means of: current, current signal shift, conductivity, resistance, impedance, phase shift, capacitance , magnetic shift and use of either direct or alternating current.
The problem with the prior art is that cells can clump as they pass through the dielectrophoresis (DEP) equipment and become contaminated.
Development of a DEP apparatus which can separate the cells and prevent contamination represents a great improvement in the field of biology and satisfies a long felt need of the biologist Development of a device and method that provides low cost 3-D DEP with simultaneous light/electrical therapy to cells while in the chamber, and at the same time avoids step down voltage fraction collection represents a great improvement in the field of medical treatment and satisfies a long felt need of patients and doctors.
Disclosure of Invention The present invention is an improved micro-electrode for DEP. The present invention is used to separate fractions of particles/cells from a heterogeneous population of particles/cells. In one example of the application, the cells may be derived from liposuctioned adipose tissue. Some novel features of this invention are:
1. Glass plates coated with indium tin oxide (ITO) to allow light therapy (i.e. laser, intense pulse light, LED) to cells for the purpose of:

a. Ablation b. Destruction c. Inactivation d. Gene expression stimulation 5 e. RNA modulation f. Cell optimization g. Cell visualization h. Collagen synthesis induction/stimulation 2. ITO surface to allow electrical conditioning of the cells.
3. The ability to switching between ITO electrodes at a proprietary frequency to levitate cell clumps and pearl chains out of the leads into the flow cell to dissociate cell clumps.
4. The ability to levitate cells into the midplane of the flow cell to allow light therapy to cells without direct damage to the planar, 2-D electrode surface.
5. The ability to test the impedance of the cell via the ITO electrodes to monitor progress of cells trapped within the cell.

6. The invention requires less processing time in that cells do not have to be eluted after successive frequency/DEP field drops.

7. The invention can use flanking border flow chambers or lane barriers to create repulsive DEP
fields which create "compartments" that keep collected cells from each array separated and confined for final elution into separate vials.
An appreciation of the other aims and objectives of the present invention and a more complete and comprehensive understanding of it may be achieved by referring to the accompanying drawings and studying the following description of the best mode of carrying out the invention.

Brief Description of Drawings Figure 1: Plan view of a prior art planar DEP electrode.
Figure 1A: Partial cross section of a prior art DEP flow chamber.
Figure 2: A conceptual representation of the instant invention having flow chambers with successively increasing dielectrophoresis fields represented by increasing shades of serially aligned rectangles in parallel to the direction of flow (arrow) and path of the cell (sphere).
Figure 3: Magnified view of planar microelectrode surface and orientation of electrode lines.
Figure 4: Conceptual representation of how different DEP fields are generated by connecting separate waveform generators to each flow chamber.
Figure 4A: Conceptual representation of an alternative embodiment. The flow cells can be connected to one waveform generator and fashioned such that the microelectrodes linearly become closer together to create the gradient DEP field.
Figure 4B is a plan view of one of the arrays in Figure 4A illustrating again that the distance between electrodes may be successively reduced.
Figure 5: Typical dimensions of the planar microelectrode array of this invention in cross section.
Figure 6: Cross sectional view of active 2-D DEP field lines represented in dotted arcs between electrodes. Cells represented in circles are trapped within the DEP fields.
Undesired parasitic cell entrapment is noted with aberrant field on far right with "X" through it.
Figure 7: Cross sectional view. Typical alternating "+" and "2 charge arrangement of planar microelectrode with overlying ITO surface electrode in off position (no charge).
Figure 8: Electrical arrangement of third or 3D electrode connected to power source and ground to waveform generator to provide DEP fields in the vertical dimension.

Figure 9: Electrical arrangement of third or 3D electrode connected to power source and ground to measure for impedance and used for diagnostic purposes such as regional saturation of cells.
Figure 10: Conceptual cross sectional view of charged/deployed electrodes in the 3D
configuration.
Figure 11: Conceptual representation of how 3D DEP fields are generated by connecting upper ITO surface to waveform generator and using both electrodes on the planar surface as one electrode. DEP fields are represented as dotted lines.
Figure 12: Conceptual representation of how a therapeutic laser used in attempting to ablate a trapped cell, will damage the nearby microelectrode if a 3D configuration or third electrode is not used.
Figure 13: Representation of how a therapeutic laser used in trying to ablate a trapped cell, will avoid damage to the nearby microelectrode if a 3D configuration or third electrode is used. At the far right the laser is incorrectly focused and delivers energy past the cell and onto the microelectrode beneath which is undesired and represented with an "X".
Figure 14: Ideal configuration with microscope (represented by the eye) above the apparatus and an intervening half mirror placed at 45 degrees to allow observation but also passage of light or therapeutic lasers or light (noted at right with the sunburst and dotted line).
Figure 15: Ideal configuration of planar electrodes placed in a sequential format relative to flow (black arrow), wherein electrodes 1 and 2 are off (missing generator symbol) and the device is in procurement or "trapping" mode.
Figure 16: The same configuration of planar electrodes as in Figure 15, however electrodes 1 and 2 are now "on" producing a cell repulsing DEP confining the cells within the central electrode. The remaining electrodes are "off" (absent generator symbol).
Elution fluid may be flooded into the central electrode region and confined cells may be collected separate from cells previously trapped on far left and far right electrode.
Figure 17: Shows an alternate configuration in which lane barriers may be skewed to allow cells to elute with only one flow through direction.

Figure 18 shows that indigo/methylene blue could be used to stain cells then run it out on the electrode and document with a video (CCD) camera.
Figure 20 shows the same "gradient" like electrode could be used with an overlying set of skewed lane barriers.
Figure 19 is an exploded view showing construction of a flow cell of this invention.
Figure 20 is a sketch of standard, straight planar array used for DEP. This is prior art.
Figure 21 is a sketch of the improved, two-dimensional, interdigitating micro electrode embodiment of this invention for DEP capture of cells.
Figure 22 is a sketch of the improved electrode embodiment of Figure 21 with a trailing, mirror electrode.
Figure 23 illustrates leading edge separation.
Figure 24 illustrates the trailing edge collection area.
Figure 25 illustrates how captured cells are held in the trailing edge collection area.
Figure 26A-7C are representational drawings of the workflow for determining the optimum electrode gap for collecting the cells of interest.
Best Mode for Carrying Out Invention Figure 1 and 1A illustrate prior art 2-D flow chambers 10. Such flow chambers 10 have a top plate 14 and a bottom plate 18. The bottom plate 18 has a microelectrode 22 adhered to its inner surface 26. This microelectrode 22 comprises a plurality of fingers 30a, 30b half of which are electrically connected together at one end 34a and, the other half of which are electrically connected together at the other end 34b. The differently connected fingers 34a, 34b alternate.
They could be described as interdigitized. In use one set of fingers 34b is connected to a positive lead and the other set 34a is connected to a negative lead. Thus cells 38 flowing through the chamber 10 in the direction D are exposed to alternating positive and negative electrical fields.

Figures 2 and 3 show that the instant invention may be made of flow chambers 42 similar to those of the prior art with microelectrodes 46 similar to those of the prior art. However, in the instant invention each successive flow chamber 42 has successively increasing dielectrophoresis field in the direction of cell flow D.
Figure 4 shows that different DEP fields can be produced by connecting separate waveform generators 50 to each microelectrode 46.
Figures 4A and 4B show that, alternatively, the flow chamber 42 can be connected to one waveform generator 50 but are fashioned such that the fingers 54 of their microelectrodes 58 linearly become closer together to create the gradient DEP field.
Figure 5 provides typical dimensions of the planar microelectrode array 58 and flow chamber 42 of this invention in cross section. The flow chamber 42 has a top plate 56 and bottom plate 60.
Figure 6 shows the 2-D DEP fields, represented by dotted lines, produced by the microelectrode arrays 58 of flow chambers 22, 42 of this and prior art inventions. Cells 38 are trapped within the DEP fields. Undesired parasitic cell entrapment is noted with aberrant field on far right with "X" through it.
Figure 7 shows typical alternating "+" and "2 charge arrangement of planar microelectrode 58 with overlying ITO electrode 62 in off position (no charge). The ITO electrode 62 is coated on a substrate 66 which is preferably glass. As is well known, ITO is electrically conductive yet transparent, which makes it the preferred material for electrodes of this invention.
Figure 8 shows the electrical arrangement of third or 3-D electrode 62 connected to power source and waveform generator 50 to provide DEP fields in the vertical dimension.
Figure 9 shows the electrical arrangement of third or 3-D electrode 62 connected to power source and ground to measure for impedance and used for diagnostic purposes such as regional saturation of cells.
Figure 10 shows the electrical configuration of charged/deployed electrodes in the 3D
configuration. In this configuration the bottom plate 60 and the ITO upper surface electrode 62 are connected together and thus have the same polarity while the fingers 54 of the microelectrode array 58 are connected together and have the opposite polarity.

Figure 11 shows the 3-D DEP fields generated by connecting the upper ITO
surface electrode 62 to the waveform generator 50 and using both electrodes on the planar surface as one electrode as shown in Figure 10. DEP fields are represented as dotted lines.
Figure 12 shows how a therapeutic laser 70 used in attempting to ablate a trapped cell 38, will 5 damage the nearby microelectrode 54 if a 2D configuration is used or when the third electrode 62 is not used.
Figure 13 shows how a therapeutic laser 70 used in trying to ablate a trapped cell 38, will avoid damage to the nearby microelectrode 54 if a 3D configuration or third electrode 62 is used. At the far right the laser 70 is incorrectly focused and delivers energy past the cell 38 and onto the 10 microelectrode 54 beneath which is undesired and represented with an "X".
Figure 14 shows the ideal configuration of the instant invention with microscope 74 above the apparatus 42 and an intervening half mirror 78 placed at 45 degrees to allow observation and passage of light or therapeutic lasers.
Figure 15 shows another ideal configuration of this invention with flow chambers 42 having planar electrodes 58 placed sequentially relative to flow D. The electrodes 58 in flow chambers 1 and 2 are off (missing generator symbol) and the device is in procurement or "trapping" mode.
Figure 16 shows the same configuration of as in Figure 15, however the electrodes 58 in flow chambers 1 and 2 are now "on" producing a cell repulsing DEP confining the cells 38 within the central flow chambers 42. Thus the electrodes 58 in chambers 1 and 2 are functioning as lane barriers 58a. The remaining electrodes 58 are "off" (absent generator symbol).
Elution fluid may be flooded into the central flow chamber 42 and confined cells 38 may be collected separately from cells 38 previously trapped on far left and far right flow chambers 42.
Figure 17 shows an alternate configuration in which lane barriers 58a may be skewed to allow cells 38 to be eluted at the ends of the lane barriers 58a.
Figure 18 shows that a flow cell 42 with a gradient DEP field could be used with an overlying set of skewed lane barriers 58a.
Figure 19 is an exploded view showing construction of a flow chamber 42 of this invention. In between the top 56 and bottom 60 plates there is a gasket 82. Two holes 86 in the top plate 56 allow for flow of cells through the flow chamber 42. Of course the inlet and outlet ports 86 could be placed elsewhere in the device ¨ for example on the ends, through the gasket 82.
As shown in Figure 20, cells 110 can clump at the center where the sample is being injected.
Flow is shown by the arrow 114. Cells clump towards the beginning of the DEP
field with a standard straight planar array 118. This causes debris and unwanted cells to become physically trapped in the clump of cells 110. In other words, clumping impedes efficient separation of debris and unwanted cells from the desired cells.
Figure 21 shows an improved micro-electrode embodiment 122 for DEP capture of cells which may be alternatively used with the previous invention disclosed in US Patent Application No.
61/525,573. Unlike the standard, straight lined, interdigitating array shown there, the 2-D, planar, interdigitating, micro-electrode array 122 shown on Figure 21 has a zigzag shape thus facilitating leading edge 132 separation and trailing edge 134 collection. By creating a wedge or angle shaped electrode 124, cells attracted to the electrode array 122 (by Fdep) will be captured, but dissipate laterally (by way of sample flow force) from the center pathway 114 of sample injection. This allows debris and unwanted cells to pass down the middle 150 unimpeded. The lateral trailing edge collection area will hold the specimen out of the midplane 150 until all sample is processed and final flush of captured cells is required.
The gap G between electrodes 124 may vary. For example the gap G at the center 136 (within the leading edge 132) may be wider than that at the trailing edge 134, such that the gap starting at the center 136 (leading edge 132) tapers as it approaches the trailing edge 134 (lateral border of array) or vice versa.
The microelectrodes 124 may be made of any suitable material such ITO (indium tin oxide), gold, copper, platinum or any electrically conducting surface and placed on a suitable substrate such as (but not limited to) glass, plastic, acrylic, PET, PMMS or PDMS.
The electrical signal applied to the array 122 may be direct or alternating current and may be modified to include standard and complex wave forms such as sine wave, triangular, stacked square and progressive sinusoidal, with or without periodic current interruption.

Figure 22 shows he same planar interdigitating micro electrode array 122 with leading edge 132 separation and trailing edge 134 collection, adjacent a second electrode 122a which is a mirror image of the first electrode 122, to allow further processing.
Figure 23 shows the leading edge 132 separation area 126 of the electrode array 122.
Figure 24 shows the trailing edge 134 collection areas 138 of the electrode array 122.
Figure 25 shows how captured cells are held in the trailing edge 134 collection area 138 and out of the midline sample flow line 150.
Figures 7A-7C are representational drawings of workflow for using a diagnostic modification of the planar 2-D array with widening gap between the electrodes to determine the optimum separation for collecting the cells 110 of interest.
A small sample of the mixed population of cells 110 is first labeled with a visibly detectable dye under microscopy. See Figure 26A. Then the sample mixture of cells 110 and labeled cells 110a are processed through a single array with multiple regions of electrode gap. In this example there are three progressively narrowing regions of 200, 100 and 50 microns. After the cells 110 are captured, an observer 130 is able to see that the pre- labeled cells 110a of interest have been captured to a specific gap width. See Figure 26B. In this example the cells of interest 110a have been captured on the region with 100 micron gap width.
The practitioner then uses a larger filter with the same gap for larger scale processing of the entire specimen 110. See Figure 26C.
The gap width can be widened to obtain a spectrum of Fdep. The cells can be labeled with pan-dye or specific flourochrome labeled Ab, which can be visualized under fluorescence microscopy to determine where that particular band of cells rests. Then the Fdep or energy within that segment using the top ITO electrode. In this way the machine can be set to the specific frequency, Vp-p and cartridge gap width.
Capacitance can be used to separate raw phases (i.e. aqueous, fatty tissue, oil) using the apparatus and method of US Patent Application Publication No. 2010/0112084 Dielectrophoresis force (Fdep) can vary as a function of distance between two electrodes or by the frequency that is generated between them. When there is positive Fdep capture force, in general, the wider the gap of the electrodes the less force is generated.
There also is a "cross over" threshold, where up to a certain point the Fdep can go from attraction to repulsion and vice versa. In other words if a certain cell population is captured with a known electrode gap (e.g.
100 microns) and a certain frequency, the same cell population can also be captured using a larger gap (e.g. 125 microns) but with a higher frequency. But the limit to this is the crossover threshold.
Antioxidants may be added to the samples to dielectrophoresis buffer.
Antioxidants may be ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotenes, alpha tocopherol (vitamin E), and ubiquinol (coenyme Q).
In addition, during processing the cells 110 may be stimulated in the wavelengths of above 760, and 400 to 450nm (in the infrared range) and light and polarized light (400-2000nm).
The electrode arrays 122 described herein can be fashioned as a syringe insert or as a submersible bioreactor.
This invention is a method and device for separating charged particles and/or cells 38 in a dielectophoretic liquid medium using gradient array dielectrophoresis. This invention provides the ability to simultaneously provide light therapy while separating the particles and/or cells 38.
Flanking microelectrodes or lane barriers 58a may be used to create repulsive DEP fields which create "compartments" that keep collected cells from each array 58 separated and confined for final elution into separate vials.
Lipoaspirate fluid may be processed by passage through serial "combs" to mechanically dissociate ADSC from the fat and tissue matrix using two bags of equal volume (similar to an IV
bag but tougher) connected together by an intervening adapter. The adapter houses a number of combs in narrowing passages One bag is filled with unprocessed lipoaspirate and compressed manually or automatically from the exterior so that the specimen is moved through the combs into the other bag. The process is repeated until the material has a smooth consistency and there is minimal resistance during fluid transfer between bags. The mixture is then transferred sterilely into a column/receptacle and allowed to settle into oil/fatty tissue/aqueous phases.

Then the phases of the lipoaspirate are separated. One way of doing this is based upon microcurrent detection wherein:
a. A small microcurrent is run between two leads at the base of the column/receptable's exit port;
b. Upon contact with the different phases (i.e. oil/fatty tissue/aqueous) differing conductivities and/or resistivities are detected; and C. A computer is programmed to detect this difference and switch a diverting valve at the base of the exit port to respective collecting receptacles/containers.
Further separation of the aqueous phase or Stromal Vascular Fraction cells and erythrocytes may be completed by free flow dielectrophoresis wherein:
a. a chamber composed of two non-conducting plates, with at least one being transparent, are separated by a small gap (0 to 5mm);
b. two electrodes are spaced apart within the gap;
C. a current is applied to the two electrodes creating a dieletrophoretic field of between 0.5 and 700 volts/cm;
d. the chamber is filled with physiologic buffer;
e. the buffer is continually renewed from the top opening of the chamber;
f. the lower chamber has an exit port with a regulator valve to adjust flow through the chamber;
g. a sample injection port is placed somewhere between the buffer ports and electrical leads;
h. the buffer port drip rates are regulated by drip regulators (similar to an iv or drip irrigation);
i. the drip rate of the sample port can be regulated in a similar fashion and rate as the buffer ports; and j. the chamber also has multiple sample collection ports wherein cells may be collected prior to exit from the buffer spill port.
This apparatus may be manufactured of disposable materials. This apparatus may be formed in a "closed system" to minimize risk of contamination and comply with GMP
5 standards.
The chamber may be modified to include:
i. a diffraction gradient to accelerate separation which is composed of microscopic barriers or posts which separate cells by size;
ii. LEDs placed in front or in back of the panels to irradiate and optimize ADSC so that 10 they are actively being separated thus minimizing processing time;
and/or iii. cooling elements added in the front and in back of the panels to prevent overheating from the electrical leads.
Additional electrodes may also be placed to compensate for dielectrophoresis field unbalance due to non-rectangular internal shape of the chamber 42. For example, the triangulated shape 15 of the upper and lower aspects of the buffer chamber causes the electric field strength to be weaker at the apical and lower pole of the electrodes. To compensate multiple electrodes with greater voltage or current can be applied towards the distal ends of the dielectrophoresis field.
Residual (non cellular fluid) may be further processed using the same apparatus as above, but this time used for separating stem cell related proteins.
To summarize, some of the features of this invention are:
1. planar micro-electrodes 58 which create a 2-D dielectrophoresis field:
2. third ITO electrodes 62 acting as cap to the flow cell 42 which create a 3-D
dielectrophoresis field 3. a waveform generator 50 of AC or DC current attached to the planar electrodes 58 4. separate waveform generator 50 of AC or DC current attached to the third ITO
electrode 62 5. a constant voltage source and a ground attached to the third ITO electrode for impedance testing 6. an overlying microscope 74 for observation 7. a half mirror 78 at 45 degree angle beneath microscope 74 8. a lateral laser 78 or IPL or LEDs optically focused to reflect off the half mirror 78 and onto middle of flow cell 42 thus not on the 2D electrodes 54;
9. a central processing unit attached to all electrodes to facilitate switching between 2-D, 3D and impedance testing mode;
10. multiple chambers 42 in series to flow, such that cells are exposed to successively increasing frequencies 11. frequencies for all electrodes 58 may be in a range from 1Hz to 100MHz.
12. chambers 42 connected to separate collection ports.
13. flow cell 42 and chamber flooded with suspension of cells/particles 38 in dielectrophoresis buffer and delivered at a constant rate to minimize cell/particle 38 shearing when cells/particles 38 are trapped on electrodes 58.
The final collection of cells/particles 38 may be done with physiologic buffer or solvent.
The microelectrodes 58 may be comprised of any suitable material such ITO
(indium tin oxide), gold, copper, platinum or any electrically conducting surface and placed on a suitable substrate such as (but not limited to) glass, plastic, acrylic, PET, PMMS or PDMS.
The electrode 58 pitch may be modified such that the space between microelectrodes 54 becomes progressively wider or narrower to create a gradient DEP field.
The electrical signal or waveform may be modified to utilize complex forms (i.e. triangular, stacked square, progressive sinusoidal) other than a standard sine wave.

The microelectrode array 58 may be modified or adapted to fit within a syringe.
The microelectrode array 58 may be modified to fit within a bioreactor. While not prior art to this invention an example of an analytical device fitted into a bioreactor is the BioSep, manufactured by Applikon Biotechnology B.V. , with headquarters at Nieuwpoortweg 12, P.O.
Box 101, 3100 AC Schiedam, The Netherlands.
Currently free flow dielectrophoresis (FFE) devices have been used for protein separation and even for separation of some micro-organisms such as bacteria and homogenized animal cells.
In an old journal article on separating bone cells at a field of 60 volts/cm an amazingly 90%
viability was found. However, nothing else could be found in the literature about using FFE for lipoaspirate processing. In addition, currently available resent FFE apparati are not disposable and do not have "Good Manufacturing Practice" standards (for clinical use) in mind. Present FFEs also require complex pumps. This invention makes the process easier and more cost efficient by using gravity and a drip like system, similar to an IV to move the buffer curtain through the apparatus. Two unique design elements to the buffer chamber are:
1. fanning ends to allow regulation down to a single point at the buffer entry and exit ports and 2. a diffusion region to accelerate separation. The advantage here is that the device used gravity and controlled drips to move the buffer curtain and sample.
The lane barriers 58a may be skewed to allow cells 38 to elute with only one flow through direction.
The distance between electrodes 54 may be successively expanded to create a gradient of DEP field. This could be used potentially as a quick and easy diagnostic test to cellularly "profile" the SVF mixture.
The same "gradient" like electrode 54 could be used with an overlying set of skewed lane barriers 58.
Indigo or methylene blue could be used to stain cells then run it out on the electrode and document with a video (CCD) camera.

Example:
3D DEP construction:
A series of three flow chambers were connected in series by flow (not electrically). Each chamber was made of an ITO coated glass plate, laser etched to produce interdigitating microelectrodes with lines having a width of 50 microns and the gap or pitch of 100 microns. A third ITO coated glass plate was used as a cap for each flow chamber thus allowing for creation of a 3D dielectrophoresis field. A barrier of 350 microns was placed between the ITO coated glass plates.
Each chamber was sealed with a polydimethylsiloxane (PDMS) gasket which circumscribed a 2.5cm x 7cm x 350 micron flow cell. Each flow cell chamber and associated electrode set was attached to separate waveform generators operated at differing frequencies noted below.
The entire apparatus was mounted on clear Plexiglas O seated atop an inverted microscope. A flat panel of LEDs to stimulate the collagen was placed directly over each flow cell when the microscope was not in use.
Sample preparation:
Infranatant from lipoaspirate was centrifuged at 300 relative centrifugal force (RCF).
The resulting pellet was washed and re-suspended in dielectrophoresis buffer containing sucrose, dextrose, tissue culture media and balanced with phosphate buffer solution (PBS) to achieve a conductivity of 30 milli Sieverts per meter (mS/m). Final concentration was approximately 1x106 cells/mL
The flow cells were flooded with DEP buffer and allowed to soak 10 minutes prior to starting the waveform generators. The 2-D waveform generator was set at 30, 60, 200 kHz for the 1st, 2nd and 3rd flow chamber electrode set respectively. The 3D
waveform generator was set to turn on every 10 minutes, for 2 minutes at X frequency, during which time the 2-D waveform generators were off. The re-suspended pellet (sample) was injected by controlled syringe pump at a rate of lcc/min. Impedance measurements were obtained at the end of each 3-D cycle.

A soak cycle in which only clean DEP buffer was injected for 30 minutes.
Direct visualization of entrapped cells was achieved light microscopy on an inverted stage.
The three chambers were eluted into separate 15mL Falcon tubes. Cell viability and counts were obtained flow cytometetry. Viability was as follows: chamber 1 95%, chamber 2 97%, chamber 3 98%; presence of CD 34 chamber 1 none, chamber 2 positive, chamber 3 none.
The method and device using dielectrophoresis to separate fractions of particles/cells from a heterogeneous population of particles/cells has been described with reference to particular embodiments. However, it should be obvious to those skilled in the art to which this invention pertains that other modifications and enhancements can be made without departing from the spirit and scope of the claims that follow.

Claims

1. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a planar, interdigitized microelectrode array on its inner surface; half of said microelectrodes being connected to a positive pole and half of said microlectrodes being connected to a negative pole; said planar, interdigitized microelectrode array being constructed to generate a 2-D dielectrophoresis field; and e) a gasket between said top and bottom plates.
the 2-D dielectrophoresis field in said distal chamber being greater than the

2-D
dielectrophoresis field in said proximal chamber;
2. An apparatus as claimed in claim 1 in which said 2-D dielectrophoresis field is produced by a wave form generator connected electronically to said planar, interdigitized microelectrode array.

3. An apparatus as claimed in claim 1 in which the spacing between adjacent microelectrodes of said planar, interdigitized microelectrode array decreases in the direction of flow; whereby said dielectrophoresis field increases in the direction of flow.

4. An apparatus as claimed in claim 1 in which said top plate is transparent.

5. An apparatus as claimed in claim 1 in which said bottom plate is transparent.

6. An apparatus as claimed in claim 1 in which said planar, interdigitized microelectrode array is transparent.

7. An apparatus as claimed in claim 1 further comprising a top electrode on the inner surface of said top plate; said top electrode connected to a wave form generator to produce a 3-D dielectrophoresis field in the vertical direction.

8. An apparatus as claimed in claim 7 in which said top electrode is transparent.

9. An apparatus as claimed in claim 7 in which said 2-D and 3-D
dielectrophoresis fields are turned on alternately.

10. An apparatus as claimed in claim 1 further comprising a microscope mounted above said top plate for viewing particles or cells within said flow chamber.

11. An apparatus as claimed in claim 1 further comprising a light source mounted above said top plate and aimed at particles or cells within said flow chamber for treatment of said particles or cells.

12. An apparatus as claimed in claim 1 further comprising a lane barrier chamber interposed in series between said proximal and distal chambers; said lane barrier comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a planar, interdigitized microelectrode array on its inner surface; said planar, interdigitized microelectrode array being constructed to generate a 2-D dielectrophoresis field; and e) a gasket between said top and bottom plates.

13. An apparatus as claimed in claim 12 in which said lane barrier is skewed in relation to direction of flow.

14. An apparatus as claimed in claim 1 wherein all components are disposable.

15. An apparatus as claimed in claim 1 in which said apparatus is constructed as a closed system.

16. An apparatus as claimed in claim 1 further comprising microscopic barriers or posts which assist in separating cells by size.

17. An apparatus as claimed in claim 1 further comprising an LED placed above said top plate.

18. An apparatus as claimed in claim 1 further comprising an LED placed below said bottom plate.

19. An apparatus as claimed in claim 1 further comprising a heat exchanger placed above said top plate.

20. An apparatus as claimed in claim 1 further comprising heat exchanger placed below said bottom plate.

21. An apparatus as claimed in claim 1 further comprising an additional electrode placed to balance said dielectrophoresis field.

22. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;

iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a planar, interdigitized microelectrode array on its inner surface; half of said microelectrodes being connected to a positive pole and half of said microlectrodes being connected to a negative pole; said planar, interdigitized microelectrode array being constructed to generate a 2-D dielectrophoresis field; and v) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

23. A method as claimed in claim 22 in which said 2-D dielectrophoresis field is produced by a wave form generator connected electronically to said planar, interdigitized microelectrode array.

24. A method as claimed in claim 22 in which the spacing between adjacent microelectrodes of said planar, interdigitized microelectrode array decreases in the direction of flow; whereby said dielectrophoresis field increases in the direction of flow.

25. A method as claimed in claim 22 in which said top plate is transparent.

26. A method as claimed in claim 22 in which said bottom plate is transparent.

27. A method as claimed in claim 22 in which said planar, interdigitized microelectrode array is transparent.

28. A method as claimed in claim 22 further comprising the step of providing a top electrode on the inner surface of said top plate; said top electrode connected to a wave form generator to produce a 3-D dielectrophoresis field in the vertical direction.

29. A method as claimed in claim 28 in which said top electrode is transparent.

30. A method as claimed in claim 28 in which said 2-D and 3-D
dielectrophoresis fields are turned on alternately.

31. A method as claimed in claim 22 further comprising the step of mounting a microscope above said top plate for viewing particles or cells within said flow chamber.

32. A method as claimed in claim 22 further comprising the step of mounting a light source above said top plate and aiming said light at particles or cells within said flow chamber for treatment of said particles or cells.

33. A method as claimed in claim 22 further comprising the step of interposing a lane barrier chamber in series between said proximal and distal chambers; said lane barrier comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a planar, interdigitized microelectrode array on its inner surface; said planar, interdigitized microelectrode array being constructed to generate a 2-D dielectrophoresis field; and e) a gasket between said top and bottom plates.

34. A method as claimed in claim 33 in which said lane barrier is skewed in relation to direction of flow.

35. A method as claimed in claim 22 in which said cells in said dieletrophoretic medium are furnished by the steps of:
a) providing two bags of equal volume;
b) providing an intervening adapter; said adapter including a plurality of narrow passages;
c) providing a plurality of combs;
d) inserting said combs in said narrow passages;
e) connecting said bags together with said intervening adapter;
f) obtaining lipoaspirate fluid;
g) filling one bag with said lipoaspirate fluid;
h) manually or mechanically compressing the exterior of said one bag so that said fluid is moved through the combs into the other bag;
i) manually or mechanically compressing the exterior of said other bag so that said fluid is moved through the combs into said one bag;
j) repeating steps h) and i) until the material has a smooth consistency and there is minimal resistance during fluid transfer between said bags;
k) sterilely transferring said material into a container having an exit port with two electrical contacts at its base and allowing it to settle into oil/fatty tissue/aqueous phases; said phases naturally having different electrical conductivities;
l) separating said phased by the steps of:
i) running a microcurrent between said electrical contacts;
ii) providing a diverting valve;

iii) providing a microprocessor electrically connected to said electrical contacts and said diverting valve; said microprocessor programmed to actuate said diverting valve at at least one conductivity;
iv) providing a second container at the exit of said diverting valve;
v) detecting said differing conductivities with said microprocessor;
vi) actuating said diverting valve by said microprocessor, whereby at least one phase is collected in said second container; said phase being said cells in said dieletrophoretic medium.

36. A method as claimed in claim 22 wherein all components are disposable.

37. A method as claimed in claim 22 in which said apparatus is constructed as a closed system.

38. A method as claimed in claim 22 further comprising microscopic barriers or posts which assist in separating cells by size.

39. A method as claimed in claim 22 further comprising an LED placed above said top plate.

40. A method as claimed in claim 22 further comprising an LED placed below said bottom plate.

41. A method as claimed in claim 22 further comprising a heat exchanger placed above said top plate.

42. A method as claimed in claim 22 further comprising heat exchanger placed below said bottom plate.

43. A method as claimed in claim 22 further comprising an additional electrode placed to balance said dielectrophoresis field.

44. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field; the spacing between adjacent fingers of said microelectrode array decreasing in the direction of flow; whereby said dielectrophoresis field increases in the direction of flow; and e) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

45. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) a transparent top electrode on the inner surface of said top plate; said top electrode connected to a wave form generator to produce a 3-D
dielectrophoresis field in the vertical direction.
c) an inlet port;
d) an outlet port;

e) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field; and f) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

46. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field;
e) a gasket between said top and bottom plates; and f) a lane barrier chamber interposed in series between said proximal and distal chambers;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

47. An apparatus as claimed in claim 46 in which said lane barrier comprises:
a) a top plate;
b) an inlet port;

c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field; and e) a gasket between said top and bottom plates.

48. An apparatus as claimed in claim 46 in which said lane barrier is skewed in relation to direction of flow.

49. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a disposable top plate;
b) a disposable inlet port;
c) a disposable outlet port;
d) a disposable bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and e) a disposable gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

50. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field; and e) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber; said apparatus being a closed system.

51. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field;
e) microscopic barriers or posts which assist in separating cells by size;
and f) a gasket between said top and bottom plates;

the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

52. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field;
e) a heat exchanger placed above said top plate; and f) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

53. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;

c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field;
e) a heat exchanger placed below said bottom plate; and f) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

54. An apparatus for separating charged particles or cells in a dielectrophoresis medium comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, connected together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber; each chamber comprising:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field;
e) an additional electrode placed to balance said dielectrophoresis field;
and f) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber.

55. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:

a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; the spacing between adjacent fingers of said microelectrode array decreases in the direction of flow; whereby said dielectrophoresis field increases in the direction of flow; and v) a gasket between said top and bottom plates;
the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

56. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;

b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) providing a transparent top electrode;
iii) attaching said transparent top electrode to the inner surface of said top plate;
iv) connecting said transparent top electrode to a wave form generator to produce a 3-D dielectrophoresis field in the vertical direction;
v) an inlet port;
vi) an outlet port;
vii) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and viii) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

57. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and v) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) providing a lane barrier;
d) connecting said lane barrier in series between said proximal and said distal chambers;
e) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
f) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

58. A method as claimed in claim 57 in which said lane barrier comprises:
a) a top plate;
b) an inlet port;
c) an outlet port;
d) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D
dielectrophoresis field; and e) a gasket between said top and bottom plates.

59. A method as claimed in claim 57 in which said lane barrier is skewed in relation to direction of flow.

60. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and v) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
a) providing two bags of equal volume;
b) providing an intervening adapter; said adapter including a plurality of narrow passages;
c) providing a plurality of combs;
d) inserting said combs in said narrow passages;
e) connecting said bags together with said intervening adapter;
f) obtaining lipoaspirate fluid;
g) filling one bag with said lipoaspirate fluid;
h) manually or mechanically compressing the exterior of said one bag so that said fluid is moved through the combs into the other bag;
i) manually or mechanically compressing the exterior of said other bag so that said fluid is moved through the combs into said one bag;
j) repeating steps h) and i) until the material has a smooth consistency and there is minimal resistance during fluid transfer between said bags;
k) sterilely transferring said material into a container having an exit port with two electrical contacts at its base and allowing it to settle into oil/fatty tissue/aqueous phases; said phases naturally having different electrical conductivities;
l) separating said phases by the steps of:
m) running a microcurrent between said electrical contacts;
n) providing a diverting valve;

o) providing a microprocessor electrically connected to said electrical contacts and said diverting valve; said microprocessor programmed to actuate said diverting valve at at least one conductivity;
p) providing a second container at the exit of said diverting valve;
q) detecting said differing conductivities with said microprocessor; and r) actuating said diverting valve by said microprocessor, whereby at least one phase is collected in said second container; said phase being said charged particles or cells in said dielectrophoresis medium;
s) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
t) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

61. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two disposable dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a disposable top plate;
ii) a disposable inlet port;
iii) a disposable outlet port;

iv) a disposable bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and v) a disposable gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

62. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for closed flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and v) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

63. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) microscopic barriers or posts;
v) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field; and vi) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

64. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field;
v) a heat exchanger placed above said top plate; and vi) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;

c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

65. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field;
v) a heat exchanger placed below said bottom plate; and vi) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;

d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;
whereby said charged particles or cells are separated.

66. A method of separating charged particles or cells in a dielectrophoresis medium comprising the steps of:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) connecting said chambers together for serial flow of said charged particles in said dielectrophoresis medium into said proximal chamber, out of said proximal chamber, into said distal chamber and out of said distal chamber;
each chamber comprising:
i) a top plate;
ii) an inlet port;
iii) an outlet port;
iv) a bottom plate; said bottom plate comprising a microelectrode array on its inner surface; said microelectrode array being constructed to generate a 2-D dielectrophoresis field;
v) an additional electrode placed to balance said dielectrophoresis field;
and vi) a gasket between said top and bottom plates the 2-D dielectrophoresis field in said distal chamber being greater than the dielectrophoresis field in said proximal chamber;
c) injecting said charged particles or cells in said dielectrophoresis medium into the inlet port of said proximal chamber;
d) causing said charged particles or cells in said dieletrophoretic medium to flow from said proximal chamber into said distal chamber;

whereby said charged particles or cells are separated.