Source: http://www.google.com/patents/US7732127?dq=5,371,548
Timestamp: 2015-12-01 22:35:54
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Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60']

Patent US7732127 - Dynamic monitoring of cell adhesion and spreading using the RT-CES system - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention includes devices and methods for dynamically monitoring cell adhesion and cell spreading. Cells are added to a microelectronic cell sensor array operably connected to an impedance analyzer. The device also includes a coating including biological molecule or organic compound capable...http://www.google.com/patents/US7732127?utm_source=gb-gplus-sharePatent US7732127 - Dynamic monitoring of cell adhesion and spreading using the RT-CES systemAdvanced Patent SearchPublication numberUS7732127 B2Publication typeGrantApplication numberUS 11/235,938Publication dateJun 8, 2010Filing dateSep 27, 2005Priority dateDec 20, 2002Fee statusPaidAlso published asCA2580548A1, EP1800312A2, EP1800312A4, US20060120204, WO2006036952A2, WO2006036952A3Publication number11235938, 235938, US 7732127 B2, US 7732127B2, US-B2-7732127, US7732127 B2, US7732127B2InventorsXiaobo Wang, Yama Abassi, Josephine Atienza, Xiao XuOriginal AssigneeAcea Biosciences, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (88), Non-Patent Citations (70), Referenced by (9), Classifications (7), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetDynamic monitoring of cell adhesion and spreading using the RT-CES system
US 7732127 B2Abstract
The present invention includes devices and methods for dynamically monitoring cell adhesion and cell spreading. Cells are added to a microelectronic cell sensor array operably connected to an impedance analyzer. The device also includes a coating including biological molecule or organic compound capable of interacting with the cell. Cell adhesion and cell mobility is determined by detecting changes in impedance and comparing impedance or cell index values between samples.
1. A method of monitoring cell adhesion or cell spreading comprising:
a) providing a device operably connected to an impedance analyzer; said device comprising:
ii) a plurality of electrode arrays and at least two connection pads positioned on said substrate, wherein each electrode array comprises at least two electrode structures, each of which comprises multiple electrode elements, further wherein the electrode structures of each electrode array have substantially the same surface area and are fabricated on a same side of said substrate, further wherein the electrode elements of each electrode structure of an electrode array are connected together to a common connection pad; and
iii) a test portion of said substrate coated with a test biological molecule or an organic compound, and a control portion of said substrate optionally coated with a biological molecule or organic compound, wherein each molecule or compound is independently selected from the group consisting of a DNA molecule, an RNA molecule, a protein, a polypeptide, an oligopeptide, an antibody, a ligand, a peptide, a receptor, one or more proteins or compounds present in the extracellular matrix (ECM), a molecule or compound capable of binding an integrin, and a cell surface receptor;
b) introducing a cell or cell population to said test portion and to said control portion;
c) performing a series of impedance measurements of said test portion and said control portion;
d) determining the change in impedance and optionally a cell index (CI) of said test portion and the change in impedance and optionally a cell index (CI) of said control portion;
e) comparing said change in impedance of said test portion to said change in impedance of said control portion or comparing said cell index (CI) of said test portion to said cell index (CI) of said control portion; and
f) determining whether cell adhesion or cell spreading on the test portion is different from that on the control portion if said comparison demonstrates a significant change in impedance.
2. The method according to claim 1, wherein said device comprises at least two test portions, wherein said biological molecule or said organic compound is positioned on said at least two test portions optionally in different concentrations, further wherein said impedance measurement is performed for each of said at least two test portions and said change in impedance and optionally said cell index (CI) is determined for each of said at least two test portions.
3. The method according to claim 1, wherein said at least two test portions are at least two test wells and said control portion is a control well, wherein said at least two test wells and said control well are perpendicularly oriented to a longitudinal axis.
4. The method according to claim 1, wherein said cell is a eukaryotic cell or said cell population is a eukaryotic cell population.
5. The method according to claim 4, wherein said eukaryotic cell is a human cell or said eukaryotic cell population is a human cell population.
6. The method according to claim 1, wherein said cell is a B-lymphocyte, T-lymphocyte or other immune cell or said cell population is a B-lymphocyte population, T-lymphocyte population or other immune cell population.
7. The method according to claim 1, wherein said series of measurements are performed at regular time intervals.
8. The method according to claim 1, wherein said series of measurements are performed at irregular time intervals.
9. The method according to claim 1, wherein said compound or biological molecule is capable of effecting cell adhesion or spreading if said comparison demonstrates a significant change in impedance.
10. The method according claim 9, wherein said biological compound or organic compound increases cell spreading or cell adhesion if change in impedance of said test portion is significantly greater than that of said control portion.
11. The method according claim 9, wherein said biological compound or organic compound is an inhibitor if change in impedance of said control portion is significantly greater than that of said test portion.
This application is a continuation in part of U.S. patent application Ser. No. 11/197,994, now U.S. Pat. No. 7,468,255 entitled, “Method for Assaying for Natural Killer, Cytotoxic T-Lymphocyte and Neutrophil-Mediated Killing of Target Cells Using Real-Time Microelectronic Cell Sensing Technology”, filed on Aug. 4, 2005, which is a continuation-in-part of U.S. patent application Ser. No. 11/055,639, now U.S. Pat. No. 7,560,269, entitled “Real time electronic cell sensing system and applications for cytotoxicity profiling and compound assays” filed on Feb. 9, 2005, which is a continuation-in-part of U.S. patent application Ser. No. 10/987,732, now U.S. Pat. No. 7,192,752, entitled “Real time electronic cell sensing system and application for cell based assays” filed on Nov. 12, 2004, which claims priority from U.S. Provisional Application 60/519,567, filed on Nov. 12, 2003. Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat. No. 7,192,752 is itself a continuation-in-part of U.S. patent application Ser. No. 10/705,447, now U.S. Pat. No. 7,470,533 filed on Nov. 10, 2003, entitled “Impedance Based Devices and Methods for Use in Assays” which claims priority to U.S. Provisional Application 60/397,749, filed on Jul. 20, 2002; U.S. Provisional Application 60/435,400, filed on Dec. 20, 2002; U.S. Provisional Application 60/469,572, filed on May 9, 2003; and is a CON of PCT application PCT/US03/22557, filed on Jul. 18, 2003. All of the applications referred to in this paragraph are incorporated by reference in their entireties herein.
This application also claims benefit of priority to U.S. Provisional Patent Application No. 60/630,131, filed on Nov. 22, 2004; U.S. Provisional Patent Application No. 60/630,071 filed on Nov. 22, 2004; U.S. Provisional Patent Application No. 60/613,872 filed on Sep. 27, 2004; U.S. Provisional Patent Application No. 60/613,749, filed on Sep. 27, 2004; U.S. Provisional Patent Application No. 60/630,809 filed on Nov. 24, 2004; U.S. Provisional Patent Application No. 60/633,019 filed on Dec. 3, 2004; 60/647,159 filed on Jan. 26, 2005; U.S. Provisional Patent Application No. 60/653,904 filed on Feb. 27, 2005; and 60/673,678 filed on Apr. 25, 2005; U.S. Provisional Patent Application No. 60/689,422 filed on Jun. 10, 2005; and is a CIP of PCT Patent Application Number PCT/US05/27943 filed on Aug. 4, 2005 and is a CIP of PCT Patent Application Number PCT/US05/27891 filed on Aug. 4, 2005. All of which are incorporated by reference in their entirety.
This application is also a continuation-in-part of U.S. patent application Ser. No. 11/198,831, entitled, “Dynamic Monitoring of Activation of G-Protein Coupled Receptor (GPCR) and Receptor Tyrosine Kinase (RTK) in Living Cells using Real-Time Microelectronic Cell Sensing Technology, filed on Aug. 4, 2005, which is herein incorporated by reference in its entirety.
Parent U.S. patent application Ser. No. 10/987,732, now U.S. Pat. No. 7,192,752 is also a continuation-in-part of U.S. patent application Ser. No. 10/705,615, now U.S. Pat. No. 7,459,303, entitled “Impedance Based Apparatuses and Methods for Analyzing Cells and Particles”, filed on Nov. 10, 2003, which claims priority to U.S. Provisional Application 60/397,749 filed on Jul. 20, 2002; U.S. Provisional Application 60/435,400, filed on Dec. 20, 2002; U.S. Provisional Application 60/469,572, filed on May 9, 2003; and is a CON of PCT application PCT/US03/22537, filed on Jul. 18, 2003. All of the applications referred to in this paragraph are incorporated by reference in their entireties herein.
Parent U.S. patent application Ser. No. 11/055,639, now U.S. Pat. No. 7,560,269 also claims priority to U.S. Provisional Patent Application No. 60/542,927 filed on Feb. 9, 2004; U.S. Provisional Application 60/548,713, filed on Feb. 27, 2004, and U.S. Provisional Application No. 60/614,601, filed on Sep. 29, 2004. All of the applications referred to in this paragraph are incorporated by reference in their entireties herein.
U.S. patent application Ser. No. 11/197,994, now U.S. Pat. No. 7,468,255 is also a continuation-in-part of PCT Patent Application No. PCT/US05/04481, filed on Feb. 9, 2005, which is a continuation-in-part of PCT Patent Application No. PCT/US04/37696, filed on Nov. 12, 2004. All of the applications referred to in this paragraph are incorporated by reference in their entireties herein.
U.S. patent application Ser. No. 11/197,994, now U.S. Pat. No. 7,468,255 also claims priority to U.S. Provisional Patent Application No. 60/598,608, filed on Aug. 4, 2004, U.S. Provisional Patent Application No. 60/630,131, filed on Nov. 22, 2004, U.S. Provisional Patent Application No. 60/689,422, filed on Jun. 10, 2005, U.S. Provisional Patent Application No. 60/598,609, filed on Aug. 4, 2004, U.S. Provisional Patent Application No. 60/613,749, filed on Sep. 27, 2004, U.S. Provisional Patent Application No. 60/647,189, filed on Jan. 26, 2005, U.S. Provisional Patent Application No. 60/647,075 filed on Jan. 26, 2005, U.S. Provisional Patent Application No. 60/660,829 filed on Mar. 10, 2005, and U.S. Provisional Patent Application No. 60/660,898 file on Mar. 10, 2005. All of the applications referred to in this paragraph are incorporated by reference in their entireties herein.
The present application relates to microelectronic devices and methods of use of to detect changes in impedance of a cell, and more specifically to microelectronic devices coated with biological molecules or organic compounds and methods of dynamically monitoring cell adhesion and cell monitoring.
The cells making up the various tissues and organs systems are held together by specific molecules that essentially serve as “biological glue” conferring shape, structure, rigidity or plasticity. During embryogenesis, these biological molecules or extracellular matrix (ECM) proteins serve as “tracks” and direct cells to the appropriate vicinity within the embryo to give rise to tissues and organ systems. ECM proteins also play a prominent role during wound healing, and are involved in directing other cellular processes such as proliferation, survival, and differentiation. Failure of cells to interact with the appropriate biological surface or molecule can be detrimental to the faith of the cells and can contribute to cancer cell metastases.
There are several methods for assessing and quantifying cellular adhesion and spreading on an ECM coated surface. The most widely used method is to apply the cells onto surfaces coated with appropriate ECM components, allow the cells to attach and adhere for a specified length of time and wash the unbound cells. The attached cells are then fixed, labeled with fluorescent reagent such as rhodamine phalloidin and visualized using an epi-fluorescent microscope or an epi-fluorescent confocal microscope. Alternatively, the cells can be labeled with a dye such as crystal violet and quantified by either counting the stained cells using a light microscope or solubilizing the stain and obtaining absorbance reading using a spectrophotometer. Cells can also be pre-labeled with a fluorescent dye for live cells such as 6-carboxyfluorescein diacetate (CFDA) and then applied to appropriate ECM-coated surface. The unbound cells are washed off and the bound cells are quantified using a plate reader. An additional method for assessing the role of integrins and other adhesion proteins is to coat different surfaces with antibodies or peptides which are specific for the various receptors and then seed the cells which are expressing the appropriate integrin receptors. The interaction of integrin receptor on the cell surface with the antibody or peptide-coated surface will allow the cells to adhere and undergo specific morphological and biological changes which can then be assessed by using cell biological techniques discussed above.
While the assays just described for assessing and quantifying cell adhesion have been informative, there are certain caveats associated with each of these assays. For example, each of the assays described are end-point assays which provide a “snapshot” of the adhesion process. All the assays involve pre-labeling or post-labeling of the cells and also involve fixation and permeabilization leading to destruction of the cell. In this application we describe a label-free real-time assay using electronic cell sensor technology (RT-CES system) which addresses some of the major limitations of the current in vitro assays for assessing the interaction of bio-molecular coated surfaces with target cells. Furthermore, because the readout is non-invasive it precludes the need for fixation and lysis of the cells and allows for acquisition of information for biological events occurring after adhesion and spreading, such as proliferation and differentiation.
The present invention includes a microlectronic cell sensor array including a non-conductive substrate, a plurality of electrode arrays positioned on the substrate, and a biological molecule or an organic compound, and optionally a control molecule or a control compound positioned on a portion of the substrate. Each electrode array includes at least two electrodes and each electrode is separated from at least one adjacent electrode by an area of non-conductive material.
In another aspect of the present invention a method of coating a microelectronic cell sensor array with a biological molecule or organic compound is provided including providing a microelectronic cell sensor array and incubating a test solution on a first portion of the electrode array and optionally a control solution on a second portion of the electrode array. The microelectronic cell sensor array may include a non-conductive substrate and a plurality of electrode arrays positioned on the substrate. Each electrode array may include at least two electrodes and each electrode may be separated from at least one adjacent electrode by an area of non-conductive material. The test solution may include a biological molecule or organic compound and the control solution may include a vehicle control and optionally a control molecule or control compound. The incubation occurs under conditions suitable for attaching the biological molecule or organic compound to the electrode array or to the nonconductive substrate.
In another aspect of the present invention, a method of monitoring cell adhesion or cell spreading is provided including providing a microelectronic cell sensor array including a test portion and a control portion, coated at least in part with a biological molecule or organic compound and operably connected to an impedance analyzer. A cell or cell population is introduced to the test portion and the control portion. A series of impedance measurements of the test portion and the control portion are performed. The change in impedance and optionally a cell index (CI) of the test portion and the change in impedance and optionally a cell index (CI) of the control portion is determined. The change in impedance of the test portion is compared to the change in impedance of the control portion or alternatively the cell index (CI) of the test portion is compared to the cell index (CI) of the control portion. Cell adhesion or cell spreading occurs if the comparison demonstrates a significant change in impedance.
In another aspect of the present invention, a method of identifying a compound or biological molecule capable effecting cell adhesion or cell spreading is provided including providing a microelectronic cell sensor array that is at least in part coated with a biological molecule or organic compound and is operably connected to an impedance analyzer, introducing a cell or cell population to a test portion and to a control portion of the microelectronic cell sensor array, performing a series of impedance measurements of the test portion and the control portion, determining the change in impedance and optionally a cell index (CI) of the test portion and the change in impedance and optionally a cell index (CI) of the control portion, comparing the change in impedance of the test portion to the change in impedance of the control portion or comparing the cell index (CI) of the test portion to the cell index (CI) of the control portion and determining cell adhesion or cell spreading is effected if the comparison demonstrates a significant change in impedance. The substrate of the microelectronic cell sensor array is coated with biological molecule or organic compound capable of supporting cell adhesion or spreading on a test portion and a control biological molecule or control organic compound on a control portion.
In another aspect of the present invention, a method of identifying an inhibitor of cell adhesion or cell spreading is provided including providing a microlectronic cell sensor array operably connected to an impedance analyzer, the microelectronic cell sensor array including a non-conductive substrate, a plurality of electrode arrays positioned on the substrate, each electrode array including at least two electrodes, and each electrode is separated from at least one adjacent electrode by an area of non-conductive material and a biological molecule or organic compound positioned on the test portion of the substrate and on a control portion of the substrate. The biological molecule or organic compound may be known to be capable of increasing cellular adhesion or cellular spreading. The method also includes preincubating a cell or cell population with a biological molecule or compound suspected of being an effector or inhibitor of cell spreading or cell adhesion defined as a test sample and preincubating a cell or cell population with a vehicle control defined as a control sample, introducing the test sample to the test portion and the control sample to the control portion, performing a series of impedance measurements of the test portion and the control portion, determining the change in impedance and optionally a cell index (CI) of the test portion and the change in impedance and optionally a cell index (CI) of the control portion, comparing the change in impedance of the test portion to the change in impedance of the control portion or comparing the cell index (CI) of the test portion to the cell index (CI) of the control portion, and determining cell adhesion or cell spreading is reduced or inhibited if the comparison demonstrates the change in impedance or cell index is greater in the control portion than the test portion.
FIG. 1 depicts a graphical representation of attachment and spreading of NIH3T3 cells on ACEA E-plates coated with fibronectin (FN) and poly-L-Lysine (PLL) and monitored by the RT-CES system. The wells of ACEA E-plates were coated with 10 μg/mL FN or with 50 μg/mL PLL for 1 hour at 37� C. The wells were washed with PBS prior to the addition of media alone for background recording. NIH3T3 cells were added at a density of 10,000 cells per well and the adhesion and spreading of the cells were monitored by the RT-CES system.
FIG. 2 depicts images demonstrating attachment and spreading of NIH3T3 cells on 16X chamber slides coated with FN and PLL. 16X chamber slides were coated with PLL and FN as described in FIG. 1. NIH3T3 cells were added to the wells and at the indicated time points the cells were fixed and stained with rhodamine-phalloidin to stain the actin cytoskeleton. The cells were visualized and imaged with an epifluorescence microscope.
FIG. 3 depicts a graphical representation of the effect of FN concentration being coated onto the ACEA E-plate on NIH3T3 cell attachment and spreading. ACEA E-plates were coated with increasing amounts of FN in the range of 0 μg/mL to 20 μg/mL. 5000 NIH3T3 cells were added to the wells and the attachment and spreading of the cells were monitored by the RT-CES system. The inset shows the average cell index of attachment at 3 hours in response to increasing amounts of FN.
FIG. 4 depicts a graphical representation of inhibition of cell attachment and spreading using the RGD containing peptides. ACEA 16X E-plates were coated with FN as described in FIG. 1. NIH3T3 cells were preincubated for 30 minutes with the indicated final concentration of the peptide GRGDS and also with the indicated concentration of the control peptide (GRADSP). The cells were added to E-plates coated with FN and the attachment and spreading of NIH3T3 cells were monitored by the RT-CES system.
FIG. 5 depicts a graphical representation of attachment and spreading of Jurkat T cells on ACEA E-plates coated with anti-CD-3 antibody. E-plates were coated with 10 μg/mL of OKT3 antibody (anti-CD-3) or a control antibody for 2 hours at room temperature. The wells were washed with PBS and then the background impedance determined using the RT-CES. 500,000 Jurkat T cells were added per well and the attachment and spreading of the cells were monitored using the RT-CES system.
FIG. 6 depicts (A) Dynamic monitoring of cell attachment and spreading on PLL and FN-coated surfaces using the RT-CES system. (B) The RT-CES measurements correlate with the extent of cell attachment and spreading using conventional phalloidin staining of the actin cytoskeleton and immunofluorescence microscopy.
FIG. 7 depicts (A) Quantitative and dynamic monitoring of cell attachment and spreading in response to increasing concentrations of FN using the RT-CES system. (B) Comparison of ACEA units of Cell Index versus manual counting of the cells for different FN concentrations at 3 hours.
FIG. 8 depicts (A) Dose-dependent inhibition of cell attachment and spreading in response to cyclic-RGD peptides, using the RT-CES system. (B) Comparison of cell attachment and spreading in response to a control peptide and cyclic-RGD peptides at three hours.
FIG. 9 depicts (A) Dynamic monitoring of the dose-dependent effect of Latriculin on NIH43T3 cell attachment and spreading on FN-coated wells, using the RT-CES system (B) Analysis of the dose-dependent effect of Latriculin on NIH3T3 cell attachment and spreading at two hours.
FIG. 10 depicts (A) Dynamic monitoring of the effect of the Src inhibitor, PP2 on BxPC3 cell attachment and spreading on FN, using the RT-CES system. (B) Comparison of the extent of cell attachment and spreading on FN in response to PP2 compared to DMSO control at two hours.
FIG. 11 depicts (A) Dynamic monitoring of cell attachment and spreading using the RT-CES system of BxPC3 cells transfected with an siRNA specific for c-Src or a control siRNA. (B) Comparison of the extent of cell attachment and spreading of BxPC3 cells transfected with c-Src siRNA or a control siRNA at two hours.
As used herein, “membrane” is a sheet of material.
As used herein, “biocompatible membrane” means a membrane that does not have deleterious effects on cells, including the viability, attachment, spreading, motility, growth, or cell division.
A “biomolecular coating” or a “biological molecule coating” is a coating on a surface that comprises a molecule that is a naturally occurring biological molecule or biochemical, or a biochemical derived from or based on one or more naturally occurring biomolecules or biochemicals. For example, a biological molecule coating can include an extracellular matrix component (e.g., fibronectin, collagens), or a derivative thereof, or can comprise a biochemical such as polylysine or polyomithine, which are polymeric molecules based on the naturally occurring biochemicals lysine and ornithine. Polymeric molecules based on naturally occurring biochemicals such as amino acids can use isomers or enantiomers of the naturally-occuring biochemicals.
As used herein, “said . . . electrodes