JP2014531199A - Gradient array dielectrophoretic separation (GrADS) with parallel phototherapy - Google Patents

Abstract

Methods and devices in which heterogeneous populations of cells or particles are introduced into a series of chambers having a continuously increasing dielectrophoretic field generated by a planar microelectrode array and are thus separated by charge and size. A transparent or translucent third electrode overlying the planar array defines the ceiling of the flow chamber and allows simultaneous introduction of phototherapy to the cells while separating the cells. The condition of having a planar electrode adjacent to the side of the capture electrode can be used to confine and retain individual encapsulated cells in the final collection and elution phase for the purpose of repulsive dielectrophoresis. . The entire process can be monitored electronically by impedance tests or visually by microscopy, in situ and in real time, without interrupting the separation process. [Selection] Figure 13

Description

  The present invention relates to the field of dielectrophoresis, and more particularly to dielectrophoresis to separate a particle / cell fraction from a heterogeneous population of particles / cells while at the same time allowing light or electrotherapy to the cells. It relates to a method and a device to be used. In one example of this application, the cells may be derived from liposuctioned adipose tissue.

Dielectrophoresis (DEP) has been used for charged particle separations since 1950 (HA Pohl, “The Motion and Precipitation of Suspensions in Diversity Electric Fields”, J. Appl. Phys. 22 (J. Appl. Phys. 22). 869-871 (1951)). The first use of this dielectrophoresis in mammalian cells was reported in 1995 in US Pat. No. 5,814,200 by Ronald Pethig.
One problem with two-dimensional (2-D) DEP is that cell aggregation can occur within the electrode, which can be further accelerated by the “pearl of chain” effect and charge accumulation. (Robert Kretschmer and Wolfgang Fritzschche Langmuir, 2004, 20 (26), 11797-11801).
Another problem with 2-D DEP is that parasitic cell capture may occur (Mario Urdaneta and Elisabeth Smela Lab Chip, 2008, 8, 550-556).
The use of three-dimensional (3-D) DEP is described in U.S. Pat. No. 1,617,978, U.S. Patent Application Publication No. 2006/0260944 (Serial No. 11 / 419,144), and U.S. Pat. No. 7,686. 934 (Serial No. 11 / 638,093). However, these documents have shown that it is difficult and complicated to manufacture electrodes.
Elution by frequency modulation is one way to separate cell fractions from heterogeneous populations. “Enrichment of putative stem cells from adipose tissue using dielectrophoresis field-flow fractionation”, Jody Vyukukal, Dayene M., et al. Vyukukal, Susanne Freyberg, Eckhard U., et al. Alt, and Peter R. C. Gascoynea, Lab Chip. 2008 Aug; 8 (8): 1386-1393 (online publication May 28, 2008, doi: 10.039 / b717043b). This is done by starting with a high “encapsulation” frequency of 200 kHz and slowly decreasing that frequency. Each unique fraction is collected as the current is reduced. However, with this method, it is difficult to adjust the timing so that different elution fractions are collected.
Currently, there is no device that provides low cost 3-D DEP while simultaneously performing light / electrotherapy on cells when in the chamber, and at the same time avoids decreasing voltage fraction collection.
The present invention relates to the inventors' earlier US patent applications 61 / 545,015, 61 / 136,932, and 12 / 578,549; This is an improvement over PCT / US09 / 6071. Non-provisional and PCT applications are listed in US Patent Application Publication No. 2010/0112084 and WIPO Publication No. Published as WO / 2010/045389. The entirety of these applications and published specifications, claims, and drawings are incorporated into this application by reference.
This improvement is due to the fact that each of the different phases (ie oil vs. fat vs. aqueous solution) detects a different electrical “signature” as it passes through the channel and activates the diversion port at the end of the channel to It also includes modifications of the prior invention apparatus to collect and separate the various phases. The electrical signature may be measured using: current, current signal shift, conductivity, resistance, impedance, phase shift, magnetic shift by either DC or AC.

US Pat. No. 5,814,200 US Pat. No. 1,617,978 US Patent Application Publication No. 2006/0260944 (Serial No. 11/419, 144) US Patent No. 7,686,934 (Serial No. 11 / 638,093) U.S. Patent Application No. 61 / 545,015 US Patent Application No. 61 / 136,932 US patent application Ser. No. 12 / 578,549 PCT patent application no. PCT / US09 / 6071 US Patent Application Publication No. 2010/0112084 WIPO Public No. WO / 2010/045389 US Patent Application No. 61 / 525,573

H. A. Pohl, “The Motion and Precipitation of Suspensions in Diversity Electric Fields”, J. Am. Appl. Phys. 22 (7), 869-871 (1951) Robert Kretschmer and Wolfgang Fritzschche Langmuir, 2004, 20 (26), 11797-11801 Mario Urdaneta and Elisabeth Smela Lab Chip, 2008, 8, 550-556 “Enrichment of putative stem cells from adipose tissue using dielectrophoresis field-flow fractionation”, Jody Vyukukal, Dayene M., et al. Vyukukal, Susanne Freyberg, Eckhard U., et al. Alt, and Peter R. C. Gascoynea, Lab Chip. 2008 August; 8 (8): 1386 to 1393 (online publication on May 28, 2008, doi: 10.1039 / b717043b)

によって与えられる。
複素誘電定数は、εが誘電定数であり、σが電気伝導率であり、ωがフィールド周波数であり、ｊが虚数である。
この方程式は、電場勾配がそれほど大きくない（例えば、電極端部に近い）場合、非常に細長い楕円に関して正確である。方程式は、形成された双極子を考慮に入れるだけであり、より高次の分極は考慮に入れない。電場勾配が大きい場合、より高次の項が関係するようになり、より高い力をもたらす。正確に言うと、時間依存性方程式は、損失があると電場と誘導双極子との間に遅れが生じるので、損失のない粒子にのみ適用される。平均すると、効果は相殺され、方程式は損失の多い粒子にも当てはまる。均等な時間平均方程式は、ＥをＥの２乗平均平方根（Ｅ_ｒｍｓ）に置き換えることによって、または正弦波電圧の場合は右辺を２で割ることによって、容易に得ることができる。
半径ｒであり複素誘電率を有する媒体中の複素誘電率の、均質な球の場合、（時間平均）ＤＥＰ力は：

である。
波括弧内の要素は、複素クラウジウス−モソッティ関数として知られ、ＤＥＰ力の全ての周波数依存性を含有する。
粒子浮揚：
粒子を、負の誘電泳動によって、平面電極アレイ上に浮揚させる。浮揚する負のＤＥＰ力、ＦＤＥＰｚ（電極アレイ上の高さを指数関数的に下降することが示されている。）、および流体力学的揚力は、沈降力、Ｆｇｒａｖに対してバランスをとり、その結果、放物線流動プロファイル内に粒子平衡位置、ｈｅｑが確立される。
中程度の流量、約１ｃｃ／時の下では、所与の粒子タイプに関する平衡高さ、ｈｅｑが：

によって表される。
従来の発明の装置には、異なる相のそれぞれ（即ち、油対脂肪対水溶液）についてそれらがチャネル内を通過するときに異なる電気「シグネチャー」を検出し、かつそのチャネルの終端で分流ポートを作動させることにより、脂肪吸引組織の種々の相を収集し分離するように、修正を行ってもよい。電気シグネチャーは：電流、電流信号シフト、導電率、抵抗、インピーダンス、相シフト、キャパシタンス、磁気シフト、および直流または交流の使用を用いて測定されてもよい。
従来技術の問題は、細胞が誘電泳動（ＤＥＰ）設備内を通過し汚染されるようになるにつれ、凝集する可能性があることである。
細胞を分離しかつ汚染を防止することができるＤＥＰ装置の開発は、生物学の分野を大幅に改善し、生物学者の長年の要望を満足させる。
チャンバ内にあるときに細胞に光線／電気療法を行うと同時に低コスト３−Ｄ ＤＥＰをもたらし、それと同時に逓減電圧画分収集を回避する、デバイスおよび方法の開発は、医学的治療の分野を大幅に改善し、患者および医師の長年の要望を満足させる。 The equations below help illustrate some of the physical principles behind methods and devices.
Dielectric force:
For a long ellipse along an electric field of radius r and length l (where r <l) having a complex dielectric constant in a medium with a complex dielectric constant, the time-dependent dielectrophoretic force is:

Given by.
In the complex dielectric constant, ε is a dielectric constant, σ is electrical conductivity, ω is a field frequency, and j is an imaginary number.
This equation is accurate for a very elongated ellipse when the electric field gradient is not very large (eg, near the electrode edges). The equation only takes into account the dipole formed, not higher order polarizations. When the electric field gradient is large, higher order terms become involved, resulting in higher forces. To be precise, the time-dependent equation applies only to particles that are not lossy because there is a delay between the electric field and the induced dipole if there is a loss. On average, the effects are offset and the equation applies to lossy particles. An equivalent time-average equation can be easily obtained by replacing E with the root mean square of E (E _rms ) or by dividing the right hand side by 2 in the case of sinusoidal voltages.
For a complex dielectric constant homogeneous sphere in a medium with a radius r and a complex dielectric constant, the (time average) DEP force is:

It is.
The element in the curly braces is known as the complex Clausius-Mossotty function and contains all the frequency dependence of the DEP force.
Particle levitation:
The particles are levitated on the planar electrode array by negative dielectrophoresis. The levitation negative DEP force, FDEPz (shown to exponentially lower the height above the electrode array), and hydrodynamic lift are balanced against the settling force, Fgrav, As a result, a particle equilibrium position, heq, is established within the parabolic flow profile.
At moderate flow rates, about 1 cc / hr, the equilibrium height, heq for a given particle type is:

Represented by
Prior art devices detect different electrical “signatures” for each of the different phases (ie oil vs. fat vs. aqueous solution) as they pass through the channel and activate the shunt port at the end of that channel By doing so, modifications may be made to collect and separate the various phases of liposuction tissue. Electrical signatures may be measured using: current, current signal shift, conductivity, resistance, impedance, phase shift, capacitance, magnetic shift, and the use of DC or AC.
A problem with the prior art is that cells can aggregate as they pass through a dielectrophoresis (DEP) facility and become contaminated.
The development of a DEP device that can separate cells and prevent contamination greatly improves the field of biology and satisfies the long-standing needs of biologists.
The development of devices and methods that deliver light / electrotherapy to cells while in the chamber while at the same time providing low cost 3-D DEP and at the same time avoiding diminishing voltage fraction collection greatly expands the field of medical therapy To satisfy the long-standing needs of patients and doctors.

The present invention is an improved microelectrode for DEP. The present invention is used to separate a particle / cell fraction from a heterogeneous population of particles / cells. In one example of the present application, the cells may be derived from liposuctioned adipose tissue. Some novel features of the present invention are as follows:
1. a. Ablation b. Destruction c. Inactivation d. Stimulation of gene expression e. RNA modulation f. Cell optimization g. Cell visualization h. A glass plate coated with indium tin oxide (ITO) to allow phototherapy (ie, laser, intense pulsed light, LED) to cells for the purpose of inducing / stimulating collagen synthesis.
2. ITO surface that allows electrical regulation of cells.
3. Ability to switch between ITO electrodes at a natural frequency to levitate cell aggregates and pearl chains from the lead to the flow cell so that the cell aggregates dissociate
4). The ability to levitate cells within the central plane of the flow cell so that phototherapy is possible for the cells without damaging the planar, 2-D electrode surface directly.
5. Ability to test cell impedance via ITO electrode to monitor the progress of cells trapped in the cell.
6). The present invention requires only a short processing time since it is not necessary to elute cells after successive frequency / DEP field drops.
7). The present invention uses adjacent boundary flow to generate a repulsive DEP field that creates a “compartment” that holds collected cells from each array that is separated and confined for final elution into individual vials. A chamber or lane barrier can be used.
An understanding of other goals and objectives of the present invention, as well as a more complete and comprehensive understanding thereof, will be realized by referring to the accompanying drawings and by studying the following description of the best mode for carrying out the invention. can do.

It is a top view of the planar DEP electrode of a prior art. 1 is a partial cross-sectional view of a prior art DEP flow chamber. FIG. Conceptual diagram of the present invention having a flow chamber that continuously increases the dielectrophoretic field represented by increasing the shadows of the rectangles arranged in series parallel to the flow (arrow) direction and the cell (sphere) path. It is. It is an enlarged view of a plane microelectrode surface and the direction of an electrode line. FIG. 6 is a conceptual diagram showing how different DEP fields are generated by connecting individual waveform generators to each flow chamber. FIG. 6 is a conceptual diagram of an alternative embodiment. The flow cell can be connected to one waveform generator and can be made such that the microelectrodes are closer together in a straight line to produce a tilted DEP field. FIG. 4B is a plan view of one of the arrays of FIG. 4A, similarly showing that the distance between the electrodes is continuously reduced. 2 is a typical dimension of a planar microelectrode array of the present invention shown in cross section. FIG. 3 is a cross-sectional view of an active 2-D DEP field line represented by ignition between electrodes. Cells represented by circles are trapped in the DEP field. Undesirable parasitic cell encapsulation is indicated by an abnormal field at the right end marked with an “X”. It is sectional drawing. A typical planar microelectrode arrangement of alternating "+" and "-" charges on which the ITO surface electrode shown in the off position overlaps (no charge) . 3 is an electrical arrangement of a third or 3D electrode connected to a power source and grounded to a waveform generator to provide a DEP field in a vertical dimension. A third or 3D electrode electrical arrangement connected to a power source and ground to measure impedance and used for diagnostic purposes such as local saturation of cells. FIG. 3 is a conceptual cross-sectional view of a charge / deployment electrode in a 3D configuration. FIG. 3 is a conceptual diagram showing how a 3D DEP field is generated by connecting the upper ITO surface to a waveform generator and using both electrodes on the plane as one electrode. The DEP field is represented as a dashed line. Schematic diagram showing how the therapeutic laser used in attempting to remove the captured cells will damage nearby microelectrodes if a 3D configuration or a third electrode is not used It is. FIG. 7 shows how a therapeutic laser used in attempting to remove captured cells avoids damage to nearby microelectrodes when a 3D configuration or a third electrode is used. . At the far right, the laser is incorrectly focused and energy is delivered past the cell to the underlying microelectrode, which is undesirably represented by an “X”. By including a microscope above the device (represented by the eye) and an intervening half mirror located at 45 degrees, not only observation is possible, but also light or therapeutic laser or light (on the right, sun and broken lines) It is a figure which shows the ideal structure which can also pass. FIG. 2 shows an ideal configuration of planar electrodes arranged in a continuous format with respect to the flow (arrow), electrodes 1 and 2 are off (no generator symbol) and the device acquires or “ It is a figure which shows the state in "capture" mode. FIG. 16 is a diagram illustrating the same planar electrode configuration as FIG. 15, but electrodes 1 and 2 are currently “on” and are generating a cell repulsion DEP that traps cells within the central electrode. The remaining electrodes are “off” (no generator symbol). The elution fluid may overflow the central electrode region and the trapped cells can be collected separately from the cells previously captured by the left and right end electrodes. FIG. 6 shows an alternative configuration where the lane barrier can be slanted to elute cells in only one flow direction. FIG. 7 shows that cells can be stained using indigo / methylene blue, then flowed over the electrodes and recorded with a video (CCD) camera, with the same “tilted” state electrodes overlaid on top of each other. FIG. 6 shows that it can be used with a set of diagonal lane barriers. It is an exploded view showing the structure of the flow cell of the present invention. A sketch of a standard, straight planar array used for DEP. This is a prior art. 2 is a sketch of an embodiment of the improved two-dimensional interdigitated microelectrode of the present invention for DEP capture of cells. FIG. 22 is a sketch of the improved electrode embodiment of FIG. 21 with a trailing edge mirror electrode. It is a figure which shows front edge separation. It is a figure which shows a trailing edge collection area. FIG. 6 shows how captured cells are retained in the trailing edge collection area. It is a figure expressing the flow of work for determining the electrode gap optimal for collecting the said cell.

1 and 1A show a prior art 2-D flow chamber 10. Such a flow chamber 10 has a top plate 14 and a bottom plate 18. The bottom plate 18 has a microelectrode 22 bonded to its inner surface 26. The microelectrode 22 includes a plurality of fingers 30a and 30b, half of which are joined together and electrically connected to one end 34a, and the other half are joined together and electrically connected to the other end 34b. The fingers 34a and 34b connected in different states are alternately arranged. These fingers can be described as interdigitated. In use, one set of fingers 34b is connected to the positive lead and the other set of fingers 34a is connected to the negative lead. Accordingly, the cells 38 flowing in the direction D in the chamber 10 are exposed to an electric field in which positive and negative are alternately arranged.
2 and 3 show that the present invention can be made with a flow chamber 42 similar to the prior art, with microelectrodes 46 similar to those of the prior art. However, in the present invention, each successive flow chamber 42 has a dielectrophoretic field that continuously increases in the direction of cell flow D.
FIG. 4 shows that different DEP fields can be generated by connecting a separate waveform generator 50 to each microelectrode 46.
4A and 4B can alternatively connect the flow chamber 42 to a single waveform generator 50, but this chamber is inclined DEP as the fingers 54 of their microelectrodes 58 are closer together in a straight line. Indicates that it is created to generate a field.
FIG. 5 shows the typical dimensions of the planar microelectrode array 58 and flow chamber 42 of the present invention by cross section. The flow chamber 42 has a top plate 56 and a bottom plate 60.
FIG. 6 shows the 2-D DEP field represented by the dashed line generated by the microelectrode array 58 of the flow chambers 22, 42 of the present invention and the prior art. Cells 38 are trapped in the DEP field. Undesirable parasitic cell encapsulation is indicated by an abnormal field at the right end marked with an “X”.
FIG. 7 shows a typical alternating “+” and “−” charge arrangement of the planar microelectrode 58 with the overlying ITO electrode 62 in the off position (no charge). Indicates a certain state. The ITO electrode 62 is coated on a substrate 66, which is preferably glass. As is well known, ITO is electrically conductive but is also transparent, making it a preferred material for the electrode of the present invention.
FIG. 8 shows the electrical arrangement of the third or 3-D electrode 62 connected to the power supply and waveform generator 50 so that the DEP field is provided in the vertical dimension.
FIG. 9 shows the electrical arrangement of a third or 3-D electrode 62 connected to power and ground to measure impedance and used for diagnostic purposes such as local saturation of cells.
FIG. 10 shows the electrical configuration of the charge / deployment electrode in a 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.
FIG. 11 shows the 3-D DEP field generated by connecting the upper ITO surface electrode 62 to the waveform generator 50 as shown in FIG. 10 and using both electrodes on the plane as one electrode. . The DEP field is represented as a dashed line.
FIG. 12 shows which therapeutic laser 70 used in attempting to remove the captured cells 38 is located on the nearby microelectrode 54 when the 2D configuration is used or when the third electrode 62 is not used. How it will be damaged.
FIG. 13 shows that the therapeutic laser 70 used in attempting to remove the captured cells 38 damages the nearby microelectrode 54 when the 3D configuration or the third electrode 62 is used. How to avoid At the far right, the laser 70 is incorrectly focused and energy is delivered to the underlying microelectrode 54 past the cell 38, which is undesirable and is represented by an “X”.
FIG. 14 shows an ideal configuration of the present invention comprising a microscope 74 above the device 42 and an intervening half mirror 78 arranged at 45 degrees to allow observation and passage of light or therapeutic laser. Indicates.
FIG. 15 shows another ideal configuration of the present invention comprising a flow chamber 42 having a planar electrode 58 arranged continuously with respect to flow D. FIG. The electrodes 58 of the flow chambers 1 and 2 are off (no generator symbol) and the device is in acquisition or “capture” mode.
FIG. 16 shows the same configuration as FIG. 15, but the electrodes 58 of the flow chambers 1 and 2 are now “on”, creating a cell repulsion DEP that traps the cells 38 within the central flow chamber 42. Thus, the electrodes 58 of chambers 1 and 2 function as lane barriers 58a. The remaining electrodes 58 are “off” (no generator symbol). The elution fluid may overflow the central flow chamber 42 and trapped cells 38 may be collected separately from the cells 38 previously captured in the left and right end flow chambers 42.
FIG. 17 shows an alternative configuration where the lane barrier 58a may be diagonal so that the cells 38 elute at the end of the lane barrier 58a.
FIG. 18 shows that a flow cell 42 with a sloped DEP field can be used with a set of overlying diagonal lane barriers 58a.
FIG. 19 is an exploded view showing the configuration of the flow chamber 42 of the present invention. Between the top plate 56 and the bottom plate 60 is a gasket 82. Two holes 86 in the top plate 56 allow cells to flow through the flow chamber 42. Of course, the inlet and outlet ports 86 can be located elsewhere on the device, such as at the end, through the gasket 82.
As shown in FIG. 20, the cells 110 may aggregate at the center where the sample is injected. Flow is indicated by arrow 114. Cells aggregate towards the beginning of the DEP field with a standard straight planar array 118. This will cause debris and unwanted cells to be physically captured by the aggregates of cells 110. In other words, aggregation prevents efficient separation of debris and unwanted cells from the desired cells.
FIG. 21 shows an improved microelectrode embodiment 122 for DEP capture of cells that may alternatively be used in conjunction with the previous invention disclosed in US patent application Ser. No. 61 / 525,573. Unlike the standard straight line interdigitated array shown there, the 2-D planar interdigitated microelectrode array 122 shown in FIG. 21 has a zigzag shape. Thus facilitating separation of the leading edge 132 and collection of the trailing edge 134. By creating a wedge-shaped or chevron-shaped electrode 124, cells that are attracted to the electrode array 122 (by F _dep ) will be captured but dissipate laterally from the central path 114 of sample injection (sample flow force). by). This allows debris and unwanted cells to pass through the center 150 unimpeded. The lateral trailing edge collection zone will hold the specimens out of the central plane 150 until all samples have been processed and a final flush of captured cells is required.
The gap G between the electrodes 124 may vary. For example, the gap G at the center 136 (within the leading edge 132) is less than the trailing edge 134 so that the gap starting at the center 136 (the leading edge 132) gradually decreases as it approaches the trailing edge 134 (the lateral boundary of the array). May be wide or vice versa.
The microelectrode 124 may be made of any suitable material such as ITO (indium tin oxide), gold, copper, platinum, or any electrically conductive surface, such as glass, plastic, acrylic, PET, PMMS, or It may be disposed on a suitable substrate such as (but not limited to) PDMS.
The electrical signals applied to the array 122 may be direct current or alternating current, and standard and composite waveforms such as sine waves, triangular waves, stacked square waves, and progressive sine waves, with or without periodic current interruption. May be modified to include.
FIG. 22 is a mirror image of the first electrode 122 for further processing, with the same planar interdigitated microelectrode array 122 having separation of the leading edge 132 and collection of the trailing edge 134. The state which has the adjacent 2nd electrode 122a is shown.
FIG. 23 shows the separation area 126 of the leading edge 132 of the electrode array 122.
FIG. 24 shows the collection area 138 of the trailing edge 134 of the electrode array 122.
FIG. 25 shows how captured cells are retained in the trailing edge 134 collection zone 138 and exit the midline sample flow line 150.
FIGS. 26A-26C depict a workflow using a planar 2D-array diagnostic modification with gaps extending between the electrodes to determine the optimal separation for collecting the cells 110 of interest. .
A small sample of the mixed population of cells 110 is first labeled with a visually detectable dye under a microscope. See FIG. 26A. The sample mixture of cells 110 and labeled cells 110a is then processed through a single array having multiple regions of electrode gaps. In this example, there are three gradually narrowing regions, 200, 100, and 50 microns. After the cell 110 is captured, the observer 130 can see that the pre-labeled cell 110a has been captured in a specific gap width. See FIG. 26B. In this example, the cell 110a is captured in a region with a gap width of 100 microns. The healthcare worker then uses a larger filter with the same gap for a larger scale treatment of the entire specimen 110. See FIG. 26C.
The gap width can be widened to obtain a wide F _dep . Cells can be labeled with a generic dye or an antibody labeled with a specific fluorescent dye, which can be visualized under a fluorescent microscope to determine where a particular cell band is located. The top ITO electrode is then used to determine the F _dep or energy within that segment. In this way, the machine can be set to a specific frequency, Vp-p, and cartridge gap width.
Capacitance can be used to separate untreated phases (ie, aqueous, adipose tissue, oil) using the apparatus and method of US 2010/0112084.
The dielectrophoretic force (F _dep ) can be varied as a function of the distance between the two electrodes or by the frequency generated between them. When there is a positive F _dep trapping force, generally less force is generated as the electrode gap widens. There is also a “crossover” threshold, ie a value that allows F _dep to go from attractive to repulsive or vice versa up to a point. In other words, if a cell population is captured at a known electrode gap (eg, 100 microns) and a frequency, the same cell population uses a larger gap (eg, 125 microns) but at a higher frequency. Can also be captured. However, this limit is the crossover threshold.
Antioxidants may be added to the sample to dielectrophoresis buffer. The antioxidant may be ascorbic acid (vitamin C), glutathione, lipoic acid, uric acid, carotene, alpha tocopherol (vitamin E), and ubiquinol (coenzyme Q).
Further, during treatment, cells 110 may be stimulated with wavelengths of 760 and above and wavelengths of 400 to 450 nm (in the infrared range), light and polarized light (400-2000 nm).
The electrode array 122 described herein can be made as a syringe insert or as an underwater bioreactor.
The present invention is a method and device for separating charged particles and / or cells 38 in a dielectrophoretic liquid medium using tilted array dielectrophoresis. The present invention provides the ability to simultaneously perform phototherapy while separating particles and / or cells 38. Adjacent microelectrodes or lane barriers 58a are used to create a repulsive DEP field that creates a “compartment” that holds the cells collected from each array 58, separated and confined for final elution in separate vials. May be.
Liposuction fluid uses two bags of similar volume connected together by an intervening adapter (similar to an IV bag, but more tough) to mechanically displace the ADSC from the fat and tissue matrix. May be processed by passing through a series “comb” to dissociate. The adapter houses several combs in a narrow passage.
One bag is filled with untreated liposuction and compressed manually or automatically from the outside so that the test specimen moves through the comb and enters the other bag. This process is repeated until the material has a smooth consistency and the resistance during fluid transfer between the bags is minimized. The mixture is then transferred to the column / receptacle in a sterile manner and allowed to settle into the oil / adipose tissue / water phase.
The lipoaspirate phase is then separated. One way to do this is based on the following microcurrent detection:
a. A small microcurrent is passed between the two leads at the base of the column / receptacle escape port;
b. Detecting different conductivity and / or resistivity when in contact with different phases (ie oil / adipose tissue / aqueous);
c. A computer is programmed to detect this difference and switch the diverter valve to the respective collection receptacle / container at the base of the escape port.
Further separation of aqueous phase or stromal vascular fraction cells and red blood cells can be terminated by the following free-flow dielectrophoresis:
a. A chamber composed of two non-conductive plates, at least one of which is transparent, is separated by a small gap (0 to 5 mm);
b. Two electrodes are spaced apart in the gap;
c. Current is applied to the two electrodes to generate a dielectrophoretic field between 0.5 and 700 volts / cm;
d. Filling the chamber with physiological buffer;
e. Constantly changing the buffer through the top opening of the chamber;
f. The lower chamber has an escape port with a regulating valve that regulates the flow in the chamber;
g. The sample injection port is located somewhere between the buffer port and the electrical lead;
h. The drip rate of the buffer port is regulated by a drip regulator (similar to iv or drip irrigation);
i. The drop rate of the sample port can be regulated in the same manner and speed as the buffer port;
j. The chamber also has a number of sample collection ports that can collect cells before exiting the buffer overflow port.
The device may be made of a disposable material. This device may be formed as a “closed system” in order to minimize the risk of contamination and comply with GMP standards.
The chamber may be modified to include the following:
I. A diffraction gradient that accelerates separation, composed of microscopic barriers or ports that separate cells by size;
II. LEDs placed in front of or behind the panel to illuminate and optimize the ADSC so as to be actively isolated and thus minimize processing time; and / or
III. A cooling element added to the front and back of the panel to prevent overheating from the electrical leads.
Additional electrodes may be placed to compensate for the dielectrophoretic field imbalance due to the non-rectangular internal shape of the chamber 42. For example, if the upper and lower aspects of the buffer chamber are triangular, the electric field strength at the apex and lower pole of the electrode will be weaker. A larger voltage or current can be applied towards the distal end of the dielectrophoresis field to compensate for multiple electrodes.
The residue (non-cellular fluid) may be further processed using the same equipment as described above, but this time it is used to separate stem cell related proteins.
In summary, some of the features of the present invention are as follows:
Planar microelectrode 58 that produces a 1.2-D dielectrophoretic field:
2.3 Third ITO electrode 62 that acts as a cap for flow cell 42 to generate a 3-D dielectrophoresis field
3. AC or DC current waveform generator 50 attached to planar electrode 58
4). Another waveform generator 50 of AC or DC current attached to the third ITO electrode 62
5. 5. Constant voltage source and ground attached to third ITO electrode for impedance test Overlying microscope 74 for observation
7). 45 degree half mirror 78 under the microscope 74
8). A side laser 78 or IPL or LED that is optically focused so that it is reflected by the half mirror 78 and enters the center of the flow cell 42, and thus other than the 2D electrode 54;
A central processing unit attached to all electrodes to facilitate switching between 9.2D, 3D and impedance test modes;
10. Multiple chambers 42 in series for flowing the cells so that they are exposed to sequentially increasing frequencies
11. The frequency of all electrodes 58 may be in the range of 1 Hz to 100 MHz.
12 Chamber 42 connected to a separate collection port.
13. Constant speed so that cell / particle 38 is filled with a suspension suspended in dielectrophoresis buffer and cell / particle 38 shear is minimized when cell / particle 38 is captured by electrode 58 Flow cell 42 and chamber delivered by
The final collection of cells / particles 38 may be performed with a physiological buffer or solvent.
The microelectrode 58 may be composed of any suitable material such as ITO (indium tin oxide), gold, copper, platinum, or any electrically conductive surface, such as glass, plastic, acrylic, PET, PMMS, or It may be disposed on a suitable substrate such as (but not limited to) PDMS.
The pitch of the electrodes 58 may be modified so that the space between the microelectrodes 54 gradually increases or decreases so that an inclined DEP field is generated.
The electrical signal or waveform may be modified to utilize a composite waveform other than a standard sine wave (ie, a triangular wave, a stacked square wave, a progressive sine wave).
The microelectrode array 58 may be modified or adapted to fit the syringe.
The microelectrode array 58 may be modified to fit the bioreactor. An example of an analytical device that is not a prior art of the present invention but is compatible with a bioreactor is headquartered by Nieuwportweg 12, P.A. O. Box 101, 3100 AC Schiedam, Applikon Biotechnology B.C. V. BioSep manufactured by
Current free flow dielectrophoresis (FFE) devices have also been used for protein separation and separation of some microorganisms such as bacteria and homogenized animal cells. In an old journal article on bone cell separation at an electric field of 60 volts / cm, surprisingly 90% survival was found. However, in this document nothing else could be found regarding the use of FFE for liposuction treatment. Furthermore, the current FFE devices currently available are not disposable and do not have “good pharmaceutical manufacturing” standards (for clinical use) in mind. The FFE also requires complex pumps. The present invention makes the process easier and more cost effective by using a gravity and drip system similar to IV to move the buffer curtain through the device. Two unique design elements of the buffer chamber are: 1. a fan-shaped end that allows the buffer entry and exit ports to be regulated to a single point; It is a scattered area that accelerates the expansion and separation. The advantage is that the device uses gravity and controls dripping to move the buffer curtain and sample.
The lane barrier 58a may be inclined to elute the cells 38 in only one flow direction.
The distance between the electrodes 54 may gradually increase to produce a slope of the DEP field. This can possibly be used as a quick and easy diagnostic test to perform a cell “profile” of the SVF mixture.
The same “tilted” electrode 54 can be used with an overlaid set of angled lane barriers 58.
Cells can be stained using indigo or methylene blue, which is then flushed to the electrodes and recorded with a video (CCD) camera.

3D DEP configuration:
A series of three flow chambers were connected in series for each flow (not electrically). Each chamber was made of a glass plate coated with ITO and etched with a laser to produce interdigitated microelectrodes using lines with a width of 50 microns and a gap or pitch of 100 microns. A glass plate coated with a third ITO was used as a cap for each flow chamber, thus creating a 3D dielectrophoresis field. A 350 micron barrier was placed between the ITO coated glass plates.
Each chamber was sealed with a polydimethylsiloxane (PDMS) gasket circumscribing a 2.5 cm × 7 cm × 350 micron flow cell. Each flow cell chamber and associated electrode set was attached to a separate waveform generator operating at the different frequencies shown below.
The entire apparatus was mounted on a transparent Plexiglas® placed directly above an inverted microscope. A flat panel of LEDs stimulating collagen was placed directly above each flow cell when the microscope was not used.
Sample preparation:
The supernatant obtained from liposuction was centrifuged at 300 relative centrifugal force (RCF). The resulting pellet was washed, resuspended in dielectrophoresis buffer containing sucrose, dextrose, tissue medium, balanced with phosphate buffer solution (PBS), and a conductivity of 30 millisieverts per meter (mS / M). The final concentration was about 1 × 10 ⁶ cells / mL.
The flow cell was filled with DEP buffer and left soaked for 10 minutes before starting the waveform generator. The 2-D waveform generator was set to 30, 60, and 200 kHz for each of the first, second, and third flow chamber electrode sets. The 3D waveform generator was set to turn on every 10 minutes, to have an X frequency for 2 minutes, and to turn off the 2-D waveform generator during that time. The resuspended pellet (sample) was infused at a rate of 1 cc / min with a controlled syringe pump. Impedance measurements were obtained at the end of each 3-D cycle.
In the immersion cycle, only clean DEP buffer was injected for 30 minutes. Direct visualization of the encapsulated cells was achieved with an optical microscope on an inverted stage. The three chambers were eluted into separate 15 mL Falcon tubes. Cell viability and number were obtained from flow cytometry. Survival rates were as follows: chamber 1 95%, chamber 2 97%, chamber 3 98%; presence of CD34 chamber 1 absent, chamber 2 present, chamber 3 absent.
Methods and devices that use dielectrophoresis to separate a particle / cell fraction from a heterogeneous population of particles / cells have been described with reference to particular embodiments. However, it should be apparent to those skilled in the art to which this invention pertains that the following can be made and other modifications and enhancements can be made without departing from the spirit and scope of the appended claims.

Claims (66)

An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate having a planar interdigitated microelectrode array on its inner surface, half of the microelectrodes connected to the positive electrode and half of the microelectrodes connected to the negative electrode, The digitized microelectrode array includes a bottom plate configured to generate a 2-D dielectrophoresis field, and e) a gasket between the top plate and the bottom plate,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber.   The apparatus of claim 1, wherein the 2-D dielectrophoretic field is generated by a waveform generator electronically connected to the planar interdigitated microelectrode array.   The apparatus of claim 1, wherein the spacing between adjacent microelectrodes of the planar interdigitated microelectrode array decreases in the direction of flow, thereby increasing the dielectrophoretic field in the direction of flow. .   The apparatus of claim 1, wherein the top plate is transparent.   The apparatus of claim 1, wherein the bottom plate is transparent.   The apparatus of claim 1, wherein the planar interdigitated microelectrode array is transparent.   The apparatus of claim 1, further comprising an upper electrode on an inner surface of the upper plate, the upper electrode connected to a waveform generator to generate a 3-D dielectrophoresis field in a vertical direction.   The apparatus of claim 7, wherein the upper electrode is transparent.   The apparatus of claim 7, wherein the 2-D and 3-D dielectrophoresis fields are alternately turned on.   The apparatus of claim 1, further comprising a microscope mounted above the upper plate to view particles or cells in the flow chamber.   The apparatus of claim 1, further comprising a light source mounted above the upper plate and for processing the particles or cells to target the particles or cells in the flow chamber. A lane barrier chamber interposed in series between the proximal chamber and the distal chamber;
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate having a planar interdigitated microelectrode array on its inner surface, wherein the planar interdigitated microelectrode array is configured to generate a 2-D dielectrophoretic field. The apparatus of claim 1 including a bottom plate and e) a gasket between the top plate and the bottom plate.   The apparatus of claim 12, wherein the lane barrier is oblique to the direction of flow.   The apparatus of claim 1, wherein all components are disposable.   The apparatus of claim 1 configured as a closed system.   The device of claim 1, further comprising a microscopic barrier or post that assists in separating cells by size.   The apparatus of claim 1, further comprising an LED disposed above the top plate.   The apparatus of claim 1, further comprising an LED disposed below the bottom plate.   The apparatus of claim 1, further comprising a heat exchanger disposed above the top plate.   The apparatus of claim 1, further comprising a heat exchanger disposed below the bottom plate.   The apparatus of claim 1, further comprising an additional electrode positioned to balance the dielectrophoresis field. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate having a planar interdigitated microelectrode array on its inner surface, half of the microelectrodes connected to the positive electrode and half of the microelectrodes connected to the negative electrode, The digitized microelectrode array includes a bottom plate configured to generate a 2-D dielectrophoresis field, and V) a gasket between the top plate and the bottom plate, in the distal chamber A 2-D dielectrophoresis field is larger than the 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated.   23. The method of claim 22, wherein the 2-D dielectrophoresis field is generated by a waveform generator electronically connected to the planar interdigitated microelectrode array.   23. The method of claim 22, wherein the spacing between adjacent microelectrodes of the planar interdigitated microelectrode array decreases in the direction of flow, thereby increasing the dielectrophoretic field in the direction of flow. .   The method of claim 22, wherein the top plate is transparent.   24. The method of claim 22, wherein the bottom plate is transparent.   24. The method of claim 22, wherein the planar interdigitated microelectrode array is transparent.   23. The method of claim 22, further comprising providing an upper electrode on an inner surface of the upper plate, the upper electrode being connected to a waveform generator to generate a 3-D dielectrophoresis field in a vertical direction. .   30. The method of claim 28, wherein the upper electrode is transparent.   30. The method of claim 28, wherein the 2-D and 3-D dielectrophoresis fields are alternately turned on.   23. The method of claim 22, further comprising placing a microscope above the upper plate to view particles or cells in the flow chamber.   23. The method of claim 22, further comprising placing a light source above the top plate and applying the light to particles or cells in the flow chamber to process the particles or cells. Interposing a lane barrier chamber in series between the proximal chamber and the distal chamber, the lane barrier comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate having a planar interdigitated microelectrode array on its inner surface, wherein the planar interdigitated microelectrode array is configured to generate a 2-D dielectrophoretic field. 23. The method of claim 22, further comprising the step of including a bottom plate and e) a gasket between the top plate and the bottom plate.   34. The method of claim 33, wherein the lane barrier is oblique to the direction of flow. The cells in the dielectrophoresis medium are:
a) preparing two bags of equal volume;
b) providing an intervening adapter including a plurality of narrow passages;
c) preparing a plurality of combs;
d) inserting the comb into the narrow passage;
e) connecting the bag together with the intervening adapter;
f) obtaining a liposuction fluid;
g) filling one of the bags with the liposuction fluid;
h) manually or mechanically compressing the outside of the one bag so that the fluid passes into the other bag through the comb;
i) manually or mechanically compressing the outside of the other bag so that the fluid enters the one bag through the comb;
j) repeating steps h) and i) until the material has a smooth consistency and resistance is minimized during fluid transfer between the bags;
k) transferring the material in a sterile manner to a container having an escape port with two electrical contacts at its base and allowing it to settle into an oil / adipose tissue / water phase, wherein the phases inherently have different electrical conductivities. Having steps,
l) said phase is
I) passing a minute current between the electrical contacts;
II) the step of providing a diversion valve;
III) providing a microprocessor electrically connected to the electrical contacts and the shunt valve, the microprocessor being programmed to operate the shunt valve with at least one conductivity;
IV) providing a second container at the outlet of the diverter valve;
V) detecting the different conductivity with the microprocessor;
VI) actuating the shunt valve by the microprocessor, thereby collecting at least one phase in the second container, the phase being separated by the step being the cells in the dielectrophoretic medium. 23. The method of claim 22, provided by:   24. The method of claim 22, wherein all components are disposable.   23. The method of claim 22, wherein the device is configured as a closed system.   23. The method of claim 22, further comprising a microscopic barrier or post that assists in separating cells by size.   24. The method of claim 22, further comprising an LED disposed above the top plate.   23. The method of claim 22, further comprising an LED disposed below the bottom plate.   23. The method of claim 22, further comprising a heat exchanger disposed above the top plate.   23. The method of claim 22, further comprising a heat exchanger disposed below the bottom plate.   24. The method of claim 22, further comprising an additional electrode positioned to balance the dielectrophoresis field. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate including a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoretic field, wherein the spacing between adjacent fingers of the microelectrode array is in the direction of flow. A bottom plate that decreases and thereby the dielectrophoretic field increases in the direction of flow; and e) a gasket between the top plate and the bottom plate;
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) a transparent upper electrode on the inner surface of the upper plate, which is connected to a waveform generator and generates a 3-D dielectrophoresis field in the vertical direction;
c) Inlet port,
d) outlet port,
e) a bottom plate including a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoretic field, and f) the top plate and the bottom plate. Including a gasket between,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate comprising a microelectrode array on its inner surface, said microelectrode array being configured to generate a 2-D dielectrophoresis field;
e) a gasket between the top plate and the bottom plate, and f) a lane barrier chamber interposed in series between the proximal chamber and the distal chamber,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. The lane barrier is
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate including a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field; and e) the top plate and the bottom plate. 48. The apparatus of claim 46, comprising a gasket between.   47. The apparatus of claim 46, wherein the lane barrier is oblique to the direction of flow. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the 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 that includes a microelectrode array on its inner surface, wherein the microelectrode array is configured to generate a 2-D dielectrophoresis field; and e) the top plate; Including a disposable gasket between the bottom plate;
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate having a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and e) between the top plate and the bottom plate Including gaskets,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger and closed system than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate comprising a microelectrode array on its inner surface, said microelectrode array being configured to generate a 2-D dielectrophoresis field;
e) a microscopic barrier or post that assists in separating cells by size, and f) a gasket between the top plate and the bottom plate,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate comprising a microelectrode array on its inner surface, said microelectrode array being configured to generate a 2-D dielectrophoresis field;
e) a heat exchanger disposed above the top plate, and f) a gasket between the top plate and the bottom plate,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate comprising a microelectrode array on its inner surface, said microelectrode array being configured to generate a 2-D dielectrophoresis field;
e) a heat exchanger disposed below the bottom plate, and f) a gasket between the top plate and the bottom plate,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. An apparatus for separating charged particles or cells in a dielectrophoretic medium, comprising at least two dielectrophoresis chambers, a proximal chamber and a distal chamber, wherein the chambers comprise the charged particles in the dielectrophoretic medium. A continuous flow is connected together such that it enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber, each chamber comprising:
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate comprising a microelectrode array on its inner surface, said microelectrode array being configured to generate a 2-D dielectrophoresis field;
e) additional electrodes arranged to balance the dielectrophoresis field, and f) a gasket between the top plate and the bottom plate,
A device in which the 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate having a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoretic field, wherein the spacing between adjacent fingers of the microelectrode array is in the direction of flow A bottom plate in which the dielectrophoresis field decreases and thereby increases in the direction of flow, and V) a gasket between the top plate and the bottom plate, and the 2-D dielectrophoresis field in the distal chamber is Being larger than the 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) To provide a transparent upper electrode,
III) attaching the transparent upper electrode to the inner surface of the upper plate;
IV) connecting the transparent upper electrode to a waveform generator to generate a 3-D dielectrophoresis field in the vertical direction;
V) Inlet port,
VI) outlet port,
VII) a bottom plate containing a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and VIII) between the top plate and the bottom plate The 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate containing a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and V) between the top plate and the bottom plate The 2-D dielectrophoresis field in the distal chamber is larger than the 2-D dielectrophoresis field in the proximal chamber;
c) providing a lane barrier;
d) connecting the lane barrier in series between the proximal chamber and the distal chamber;
e) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
f) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. The lane barrier is
a) Top plate,
b) Inlet port,
c) outlet port,
d) a bottom plate that includes a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and e) the top plate and the bottom plate. 58. The method of claim 57, comprising a gasket in between.   58. The method of claim 57, wherein the lane barrier is oblique to the direction of flow. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate having a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and V) of the top plate and the bottom plate A 2-D dielectrophoresis field in the distal chamber is larger than a 2-D dielectrophoresis field in the proximal chamber;
a) preparing two bags of equal volume;
b) providing an intervening adapter including a plurality of narrow passages;
c) preparing a plurality of combs;
d) inserting the comb into the narrow passage;
e) connecting the bag together with the intervening adapter;
f) obtaining a liposuction fluid;
g) filling one of the bags with the liposuction fluid;
h) manually or mechanically compressing the outside of the one bag so that the fluid passes into the other bag through the comb;
i) manually or mechanically compressing the outside of the other bag so that the fluid enters the one bag through the comb;
j) repeating steps h) and i) until the material has a smooth consistency and resistance is minimized during fluid transfer between the bags;
k) The step of sterilizing the material into a container having an escape port with two electrical contacts at its base and allowing it to settle into an oil / adipose tissue / water phase, the phases having inherently different electrical conductivities Step,
l) said phase is
m) a sub-step of passing a minute current between the electrical contacts;
n) a sub-step of providing a shunt valve;
o) a sub-step of providing a microprocessor electrically connected to the electrical contact and the shunt valve, wherein the microprocessor is programmed to operate the shunt valve with at least one conductivity;
p) a sub-step of providing a second container at the outlet of the diversion valve;
q) a sub-step of detecting said different conductivity with said microprocessor; and r) a sub-step of operating said shunt valve with said microprocessor, thereby collecting at least one phase in said second vessel; Separating the phases by sub-steps being the charged particles or cells in the dielectrophoretic medium s) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
t) A method comprising flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber, whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two disposable dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Disposable top plate,
II) Disposable inlet port,
III) Disposable outlet port,
IV) a disposable bottom plate that includes a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and V) the top plate and the bottom plate A 2-D dielectrophoresis field in the distal chamber is larger than a 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the closed flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Connecting the chambers together,
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate having a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and V) of the top plate and the bottom plate A 2-D dielectrophoresis field in the distal chamber is larger than a 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) microscopic barriers or posts,
V) a bottom plate that includes a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field, and VI) between the top plate and the bottom plate A 2-D dielectrophoresis field in the distal chamber is larger than a 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate comprising a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field;
V) a heat exchanger disposed above the top plate, and VI) a gasket between the top plate and the bottom plate, wherein the 2-D dielectrophoresis field in the distal chamber is the proximal chamber A step that is larger than the 2-D dielectrophoresis field in
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate comprising a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field;
V) a heat exchanger disposed below the bottom plate, and VI) a gasket between the top plate and the bottom plate, wherein the 2-D dielectrophoresis field in the distal chamber is the proximal chamber A step that is larger than the 2-D dielectrophoresis field in
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated. A method for separating charged particles or cells in a dielectrophoretic medium, comprising:
a) providing at least two dielectrophoresis chambers, a proximal chamber and a distal chamber;
b) the chamber such that a continuous flow of the charged particles in the dielectrophoretic medium enters the proximal chamber, exits the proximal chamber, enters the distal chamber, and exits the distal chamber; Are the steps to connect together
Each chamber
I) Top plate,
II) Inlet port,
III) Outlet port,
IV) a bottom plate comprising a microelectrode array on its inner surface, the microelectrode array being configured to generate a 2-D dielectrophoresis field;
V) additional electrodes arranged to balance the dielectrophoresis field, and VI) a gasket between the top plate and the bottom plate, the 2-D dielectrophoresis field in the distal chamber is Being larger than the 2-D dielectrophoresis field in the proximal chamber;
c) injecting the charged particles or cells in the dielectrophoretic medium into the inlet port of the proximal chamber;
d) flowing the charged particles or cells in the dielectrophoretic medium from the proximal chamber to the distal chamber;
A method whereby the charged particles or cells are separated.

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